Hohagen et al. BMC Developmental Biology  (2015) 15:19 
DOI 10.1186/s12861-015-0068-7METHODOLOGY ARTICLE Open AccessAn optimised whole mount in situ hybridisation
protocol for the mollusc Lymnaea stagnalis
Jennifer Hohagen, Ines Herlitze and Daniel John Jackson*Abstract
Background: The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental
and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through
inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model
organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated
for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded,
labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using
colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final
result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in
situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic
stage within a species.
Results: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both
whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity
for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably
significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent
WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which
eliminates this signal.
Conclusions: This method that we present here will be of value to investigators employing L. stagnalis as a model
for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and
brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.
Keywords: Whole mount in situ hybridisation, Mollusc, Lymnaea stagnalis, Gene expression, Evolution, DevelopmentBackground
Analysing how spatial and temporal developmental gene
expression profiles evolve is a powerful strategy for un-
derstanding how morphological diversity can be gener-
ated. The most commonly employed technique for the
study of spatial gene expression in a given tissue or de-
velopmental stage is in situ hybridisation (ISH), often
applied to whole embryos or larvae as whole mount in
situ hybridisation (WMISH). WMISH provides informa-
tion about the timing and localisation of a gene’s expres-
sion in a developing embryo or larva, and can be used to
characterise and identify cell types, tissues or organs
within the whole organism and to make inferences about* Correspondence: djackso@uni-goettingen.de
Department of Geobiology, Geosciences Centre, Georg-August University of
Göttingen, Goldschmidtstrasse 3, 37077 Göttingen, Germany
© 2015 Hohagen et al.; licensee BioMed Centr
Commons Attribution License (http://creativec
reproduction in any medium, provided the or
Dedication waiver (http://creativecommons.or
unless otherwise stated.their function and evolutionary history [1-3]. Unfortu-
nately, the technique can be challenging, especially when
applied to an organism for which there is little knowledge
regarding the multifarious conditions that optimise the
balance between WMISH signal intensity and the preser-
vation of morphological integrity, two often conflicting
requirements. WMISH experiments on embryos can be
further challenged by changes in the biochemical and bio-
physical properties of the developing tissues during onto-
genesis. Thus, the procedure often needs to be adapted for
distinct developmental stages within a species.
From an evo-devo perspective, the pulmonate fresh-
water gastropod Lymnaea stagnalis (Linnaeus, 1758) is a
representative of a significantly understudied group of
animals, the Spiralia/Lophotrochozoa. Primarily due to
its availability and ease of culture, L. stagnalis was onceal. This is an Open Access article distributed under the terms of the Creative
ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
iginal work is properly credited. The Creative Commons Public Domain
g/publicdomain/zero/1.0/) applies to the data made available in this article,
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 2 of 13a much used model for studying molluscan development
[4-6], and is currently employed as a model for studies
focused on various biological processes including the
establishment of chirality [7], the evolution of shell for-
mation [8] and ecologically regulated development [9].
However, L. stagnalis possesses certain traits that repre-
sent technical challenges to WMISH. First, L. stagnalis
embryos develop individually within egg capsules filled
with a fluid that serves a nutritive function and is upta-
ken by pinocytosis during development [10-12]. This
viscous intra-capsular fluid, which consists of a com-
plex mixture of ions, polysaccharides, proteoglycans and
other polymers [13], can be seen to stick to the embryo
following decapsulation, and likely interferes with any
WMISH procedure. Second, from 52 hours post first
cleavage onwards the first insoluble material associated
with shell formation is secreted [8]. This material non-
specifically binds some nucleic acid probes and gener-
ates a characteristic background signal. This phenomena
is not restricted to L. stagnalis but can be observed in
larvae of other gastropods (our unpublished data), bi-
valves, scaphopods and polyplacophoran molluscs (pers.
comm. Tim Wollesen). Finally, L. stagnalis embryos and24 h 48 h 72 h
A B C
F G H
st
K L M
25 h 50 h 70 h
24 h 48 h 72 h
bp
pt pt
apap
pt
Figure 1 Overview of the early larval development of L. stagnalis. Dur
drastic changes in size (A-E, images are to the same scale shown in E), tissue
are the positions of the apical plate (ap), the eye (ey), the foot lobe (fl) or foot
and the blastopore (bp) or stomodaeum (st). All ages are indicated in hours p
representing 50 μm. I, J, N and O are lateral views with scale bars of 100 μm.
about the vertical axis for consistency of presentation.larvae undergo significant morphometric and biophysical
changes in the characteristics of their tissues during the
first days of development (Figure 1). Previously de-
scribed WMISH protocols for larvae of L. stagnalis pro-
duced WMISH signals with low signal to noise ratios,
making some previously reported gene expression pat-
terns difficult to interpret [14-16].
In order to achieve consistent WMISH signals in L.
stagnalis larvae with maximum signal to noise ratios, we
have systematically compared the influence of a variety
of chemical and enzymatic pre-hybridisation treatments
previously reported to address each of these challenges.
We first evaluated the effect of the mucolytic agent N-
acetyl-L-cysteine (NAC) in order to assess the possibly
negative influence of the intra-capsular fluid on WMISH
in L. stagnalis. A treatment with NAC has been shown
to improve WMISH signal intensity in the platyhelminth
flatworm Schmidtea mediterranea, presumably by de-
grading the mucosal layer surrounding the animal and
thereby increasing accessibility of the probe to the tissue
[17]. WMISH signal quality was also improved in S.
mediterranea through the use of the reducing agent di-
thiothreitol (DTT) and the detergents sodium dodecyl96 h 120 h
D E
I J
st
eyN O
100 h 120 h
96 h 120 h
mm
f
st
pt
aps
f
mm
st
ey
ap
ing the first five days of development, embryos of L. stagnalis undergo
composition (F-J) and form all main larval structures (K-O). Indicated
(f), the developing mantle margin (mm), the prototroch (pt), the shell (s)
ost first cleavage (h). F-H and K-M are ventral views with scale bars
The scale bar in panel E is 500 μm. Panels I, J, N and O are reflected
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 3 of 13sulfate (SDS) and NP-40, a treatment referred to by
Pearson et al. as ‘reduction’ [17]. An alternative permeabi-
lising treatment solely utilising SDS is commonly employed
in WMISH protocols for a variety of animals such as the
platyhelminth S. mediterranea [18] or the arthropod Par-
hyale hawaiensis [19,20]. Here we assess the impact of dif-
ferent combinations of these and other standard WMISH
treatments (storage, enzymatic permeabilisation by Protein-
ase K (Pro-K), acetylation) on the strength and consistency
of the WMISH signal across early developmental stages of
L. stagnalis. We also systematically evaluated the effects of
the Alkaline Phosphatase (AP) -conjugated anti-DIG anti-
body concentration, the composition of the colour detec-
tion solution and different probe preparation approaches.
We have performed these experiments with a selection of
three genes, which can be reasonably assumed to have dif-
ferent levels of expression: beta tubulin, and the transcrip-
tion factors engrailed and COE (collier/olfactory-1/early B
cell factor). We also demonstrate the presence of a tissue-
specific background stain, which can be abolished by treat-
ment with triethanolamine (TEA) and acetic anhydride
(AA). The optimised WMISH method we present here will
allow for future molecular studies to be performed on a
wide range of developmental processes within L. stagnalis.
Methods
A detailed list of all solutions used can be found at the
end of this section. If not otherwise indicated, all steps
were carried out at room temperature.
Cultivation of adult L. stagnalis and preparation of
embryos
Laboratory cultures derived from adult L. stagnalis col-
lected from the Northeimer Seenplatte, Germany, from a
pond on the North campus of the University of Göttingen,
Germany, and from Nottingham, U.K. and were kept in
standard tap water at 25°C, under a 16:8 light dark regime
and fed ad libidum with lettuce and a variety of other veg-
etables. Under this regime adult snails lay egg masses year
round. Egg masses of diverse ages were collected and
grouped into three developmental time windows: from
one to two days post first cleavage (dpfc), from approxi-
mately two to three dpfc and from three to five dpfc. In-
dividual egg capsules were freed from the surrounding
jelly by rolling them over moist filter paper. Embryos were
released from their egg capsules by manual dissection
using forceps and mounted needles. In order to minimise
experimental error, embryos for each experiment were
pooled and processed up to a point when experiment-
specific treatments were applied.
NAC treatment
Freshly dissected embryos were immediately incubated
in NAC solution. The duration and concentration of thistreatment were age-dependent. Embryos ranging from
two to three dpfc were treated for five minutes with
2.5% NAC, and samples between three and six dpfc were
treated with 5% NAC twice for five minutes each. All
samples were then immediately fixed for 30 minutes in
4% paraformaldehyde (PFA) in PBS.Fixation
All samples were transferred into freshly prepared 4%
(w/v) PFA in 1X PBS and incubated for 30 minutes at
room temperature. The fixative was removed by one
wash for five minutes in 1X PBTw. Samples were then
subjected to a treatment with SDS.SDS treatment
Following fixation, all samples were washed once in
PBTw for five minutes and then incubated in 0.1% SDS,
0.5% SDS or 1% SDS in PBS for ten minutes at room
temperature. Following the SDS treatment, samples were
rinsed in PBTw and dehydrated through a graded etha-
nol (EtOH) series in PBTw; one wash in 33% (v/v)
EtOH, one wash in 66% (v/v) EtOH and two washes in
100% EtOH, each wash lasting five to ten minutes. All
samples were then stored at −20°C.Reduction
Following fixation and one five minutes wash in PBTw,
embryos between two and three dpfc were treated with
0.1X reduction solution for ten minutes at room
temperature. Embryos between three and five dpfc were
incubated for ten minutes in preheated 1X reduction so-
lution at 37°C. All samples were carefully inverted once
during this time. We found all samples to be extremely
fragile in this solution and should be handled with care.
After removal of the reduction solution, all samples were
briefly rinsed with PBTw before being dehydrated
through a graded EtOH series; one wash in 50% (v/v)
EtOH, two washes in 100% EtOH, each wash lasting five
to ten minutes. All samples were then stored at −20°C.
Note: this treatment replaces the SDS treatment de-
scribed above.RNAse treatment in order to investigate the source of
non-specific WMISH staining
NAC-treated samples were fixed and dehydrated as de-
scribed above and stored at −20°C. Samples were then
rehydrated through a graded EtOH series into PBTw and
then incubated for 30 minutes at 37°C in 10 μg/ml and
100 μg/ml RNAse A (Sigma, #R5503) in 2X SSC. Samples
were then washed five times in PBTw for 5 minutes each
before proceeding with Proteinase K digestion.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 4 of 13Protein digestion with Proteinase K (Pro-K)
Following fixation, dehydration, storage at −20°C and
any additional treatments (NAC, reduction or SDS),
samples were rehydrated through a graded EtOH series
into PBTw. Embryos were then treated with an age-
dependent concentration of Pro-K (Carl Roth, #7528)
for ten minutes at room temperature. The regimes ul-
timately employed are the culmination of a more ex-
haustive series of trials using a greater range of Pro-K
concentrations (0 to 50 μg/ml Pro-K). Embryos between
one and two dpfc were incubated in concentrations of
Pro-K ranging from 1–15 μg/ml, embryos between two
and three dpfc in concentrations of Pro-K ranging from
5–20 μg/ml and older embryos (between three and five
dpfc) were treated at concentrations between 5 μg/ml
and 40 μg/ml. Pro-K activity was stopped by two five
minutes washes in 2 mg/ml glycine. All samples were
then briefly rinsed in PBTw.
Triethanolamine + acetic anhydride (TEAAA) treatment
Samples were transferred into a 1% (v/v) solution of
triethanolamine (TEA) in PBTw and incubated for five
minutes. This step was then repeated. This solution was
then replaced with a solution of 1% TEA + 0.3% (v/v)
acetic anhydride (TEAAA) in PBTw. This step was re-
peated for some samples. All samples were then washed
once with PBTw, postfixed for 15 to 20 minutes in 4%
PFA in PBTw, and washed three times with PBTw be-
fore being transferred into an Intavis In situ-Pro robot
for all subsequent hybridisation, antibody incubation
and washing steps.
Riboprobe synthesis
Primers designed to amplify fragments of beta tubulin,
engrailed and COE were designed from 454 and Illumina
RNASeq data (see Additional file 1 for all primer se-
quences). These PCR products were cloned into vectors
containing T7 and SP6 promotor sites and verified by
Sanger sequencing. These fragments were then amplified
from plasmid DNA using M13 primers, and purified using
the QIAGEN QIAquick Gel Extraction Kit. Antisense
riboprobes were synthesised using Promega reagents in a
10 μl-reaction containing 1X reverse transcription buffer,
10 mM Dithiothreitol, 1X Digoxigenin RNA labeling Mix
(Roche, #11277073910), 0.25 - 0.5 volume PCR template
and 20 Units of the appropriate RNA polymerase (SP6 or
T7; Promega, #P108 or #P207). Probe synthesis reactions
were carried out at 37°C for two to four hours. For beta
tubulin, a 702 bp long internal fragment was used for
riboprobe synthesis. For engrailed, a 929 bp internal frag-
ment partially covering the homeobox domain was used.
The riboprobe against COE was generated from a 1626 bp
long internal fragment covering the DNA binding domain
and the TIG/IPT domain. All riboprobes were purified byprecipitation using 0.1 volume of 3 M sodium acetate
pH 5.2 and 3 volumes of absolute EtOH for 15 minutes,
and subsequently centrifuged for 15 minutes at 16,000
RCF. All precipitation steps were carried out at room
temperature. The resulting pellets were washed once in
75% EtOH, dried and dissolved in 10 μl water at 55°C.
After quantification using a Nanodrop, 500 ng of ribop-
robe was denatured in 95% deionised formamide at 75°C
for 10 minutes and qualitatively assessed by agarose gel
electrophoresis. The remaining riboprobe solution was ad-
justed to a final concentration of 300 ng/μl using deio-
nised formamide. In order to assess the affect of probe
hydrolysis on WMISH signal, some riboprobes were also
hydrolysed as described by [17].
Probe hybridisation and antibody binding
All samples were incubated in hybridisation buffer for
15 minutes at room temperature before being heated to
the hybridisation temperature of 55°C. The hybridisation
buffer was then exchanged and incubated for an add-
itional two hours. Each riboprobe in hybridisation buffer
was denatured for ten minutes at 75°C and aliquoted
into individual hybridisation reaction tubes for subse-
quent use in the robot. The hybridisation buffer on all
samples was replaced by the riboprobe in hybridisation
buffer and allowed to hybridise for 16 hours at 55°C
using the following optimised concentrations of ribo-
probes: beta tubulin 100, 150 or 200 ng/ml; engrailed
500 ng/ml; COE 100 ng/ml or 300 ng/ml. Unbound probe
was washed out with three washes in 4X wash buffer for
15 minutes each, three washes in 2X wash buffer for 15
minutes each, three washes in 1X wash buffer for 15
minutes each and one wash in 1X SSC + 0.1% Tween for
15 minutes, all performed at 55°C. Samples were then
allowed to cool to room temperature and then washed
twice in 1X SSC + 0.1% Tween for 15 minutes each. Two
washes in maleic acid buffer (MAB) pH 7.5 were then per-
formed for ten minutes each. All samples were then
cooled to 10°C and incubated for three hours and 30 min
in pre-cooled 2% block solution (Roche, #11096176001) in
MAB with one exchange. Block solution was then re-
placed by block solution containing a 1/10,000 dilution of
anti-DIG antibody conjugated to Alkaline Phosphatase
(Roche, #11093274910) and incubated for five hours
followed by a renewal of this solution and a further five
hours incubation, all at 10°C. Samples were then allowed
to warm to room temperature and unbound antibody was
removed by 15 washes with PBTw for ten minutes each.
Colour development and postprocessing
For colour development, samples were transferred into
1X Alkaline Phosphatase buffer with 0.1% (v/v) Tween
20 (APTw) and incubated with two ten minutes washes
at room temperature. The 1X APTw buffer was replaced
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 5 of 13by the detection buffer and colour development was per-
formed in the dark. Fluorescent signals were developed
using the SIGMA FAST™ Fast Red TR/Napthol AS-MX
Alkaline Phosphatase Substrate (Sigma, #F4648), prepared
according to the manufactorer’s recommendations. All re-
actions were stopped by replacing the colour substrate so-
lution with three five minutes washes in PBTw each,
followed by two five minutes washes in 0.1 M Glycine pH
2. Samples for direct comparisons were stopped at the
same time. After three further five minutes washes in
PBTw, samples were postfixed in 4% (v/v) PFA in PBTw
for at least two hours at room temperature or over night
at 4°C. The fixative was removed by two five minutes
washes with PBTw, followed by two washes with pre-
warmed deionised water for each ten minutes at 37°C.
Embryos were then dehydrated through a graded EtOH
series (33%, 66%, twice with 100%) and stored at −20°C.Imaging
Prior to imaging samples were rehydrated through a
graded EtOH series in PBTw (66%, 33%, twice with PBTw)
and cleared at 4°C over night in 60% (v/v) glycerol. For
“bulk” imaging (where a single image of tens of embryos
gives an impression of the consistency of a given treat-
ment) embryos were mounted in a 96 well plate with
U-shaped bottom and imaged under a Zeiss stereo Discov-
ery V8 microscope running Zeiss camera software Axio
Vision Rel. 4.7. For images of individual embryos, samples
were mounted on glass slides and photographed using a
Zeiss Axio Imager Z1 microscope running Zeiss camera
software Axio Vision Rel. 4.8. Images of all samples were
captured using automatic settings for exposure and white
balance. Images of individual embryos were also captured
at different focal planes some of which were projected
using Macnification version 2.0.1. All images were edi-
ted in Adobe Photoshop CS3 version 10.0.1 to achieve
the optimal visual representation of each WMISH treat-
ment and to facilitate qualitative comparisons. Each
image was linearly adjusted for brightness, contrast and
colour balance using the automatic function. These ad-
justments were applied to every pixel in each image
and did not obscure, eliminate, or misrepresent any
information.
Fluorescence detection was performed using a Zeiss
LSM 510 Meta with the following microscope setup:
HeNe 543 laser, HFT 488/543/633; NFT490; LP560.
Both individual images and stacks were captured using
the following settings: laser power of 2.9%; pinhole of
59 μm; amplifier gain of 1; amplifier offset and gain
adjusted to the sample brightness; stack size of 1024 ×
1024; scan speed and number of scans 7 and 4 respect-
ively. For individual images the stack size was 2048 ×
2048.Solutions
1X PBS (phosphate buffered saline): 0.1 volume of
10X PBS stock (1.37 M NaCl; 27 mM KCl; 100 mM
Na2HPO4.2H2O; 20 mM KH2PO4)
1X PBTw (phosphate buffered saline + Tween 20):
10% (v/v) of 10X PBS stock; 0.1% (v/v) Tween-20
2.5% NAC (N-acetyl cysteine): 50% (v/v) of 5% (w/v)
NAC in 1X PBS
4% PFA (paraformaldehyde): 25% (v/v) of 16% (w/v)
PFA pH 7–8; 1X PBS or 1X PBTw
0.5% SDS (sodium dodecyl sulfate): 2.5% (v/v) of
20% (w/v) SDS; 1X PBS
0.1X reduction solution: 0.1% (v/v) Tergitol (NP40);
0.05% (v/v) sodium dodecyl sulfate (SDS); 5 mM dithio-
threitol (DTT)
1X reduction solution: 1% (v/v) Tergitol (NP40);
0.5% (v/v) SDS, 50 mM DTT
33% EtOH (ethanol): 33% (v/v) volume of absolute
EtOH in PBTw
66% EtOH (ethanol): 66% (v/v) volume of absolute
EtOH in PBTw
Pro-K (Proteinase K): Diluted from 10 mg/ml stock
using PBTw
2 mg/ml glycine pH 2: Diluted from 100 mg/ml stock
using PBTw
TEA (triethanolamine): 1% (v/v) TEA diluted in 1X
PBTw
TEAAA (triethanolamine + acetic anhydride): 1%
(v/v) TEA; 0.3% (v/v) acetic anhydride diluted in 1× PBTw
Hybridisation buffer: 25% (v/v) 20X SSC stock (3 M
NaCl; 0.3 M trisodium citrate dihydrate); 5 mM ethylene
diamine tetra-acetic acid (EDTA) from 500 mM stock
pH 8.0; 0.5 volume deionised formamide; 100 μg/ml
Heparin from 100 mg/ml stock; 0.1% (v/v) Tween-20;
1X Denhardt’s from 100X stock (2% (m/v) Ficoll type
400; 2% (w/v) polyvinylpyrrolidone K30; 2% (w/v) bovine
serum albumin); 100 μg/ml single-stranded salmon
sperm DNA from 10 mg/ml stock
4X wash: 20% (v/v) 20X SSC stock; 50% (v/v) formam-
ide; 0.1% (v/v) Tween-20
2X wash: 10% (v/v) 20X SSC stock; 50% (v/v) formam-
ide; 0.1% (v/v) Tween-20
1X wash: 5% (v/v) 20X SSC stock; 50% (v/v) formam-
ide; 0.1% (v/v) Tween-20
MAB (maleic acid buffer): 0.1 M maleic acid from 1
M stock pH 7.5; 0.15 M NaCl from 5 M stock
Block solution: 2% (v/v) block from 10% (w/v) stock
in MAB
Antibody solution: AP-conjugated anti-DIG fab frag-
ments diluted 1/10,000 in block solution
1X APTw (Alkaline Phosphatase buffer + Tween
20): 20% (v/v) 5X AP buffer stock (0.5 M Tris pH 9.5
from 1 M stock; 0.5 M NaCl from 5 M stock); 0.1% (v/v)
Tween-20
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 6 of 13Colour detection buffer: 1X APTw; 50 mM MgCl2
from 1 M stock; 450 μg/ml NBT from 100 mg/ml stock
in DMF; 175 μg/ml BCIP from 50 mg/ml stock in water
Colour detection buffer + PVA (polyvinyl alcohol):
1X APTw; 50 mM MgCl2 from 1 M stock; 450 μg/ml
NBT from 100 mg/ml stock in DMF; 175 μg/ml BCIP
from 50 mg/ml stock in water; all diluted in 10% (w/v)
PVA in water
Fluorescent colour detection buffer: 0.1 M Tris;
1 mg/ml Fast Red TR; 0.4 mg/ml Napthol AS-MX;
0.15 mg/ml Levamisol; final pH 7.9-8.5
Stop solution: 0.1 M glycine pH 2.2 from 1 M stock;
0.1% (v/v) Tween-20.
PFA: 4% (v/v) PFA in 1x PBTw
60% glycerol: 60% glycerol (v/v) in water
Results and discussion
Previously described WMISH protocols for molluscan
embryos and larvae did not yield consistent or satisfactory
WMISH signals in L. stagnalis [14,21,22]. Therefore, we
focused on a few key steps of sample preparation we be-
lieved to cause background, weak WMISH signals and
non-specific staining. Note that in this work we did not
explore the effect of hybridisation temperature on the final
result. Initial experiments with the probes used in the
present study revealed that 55°C produced consistent and
acceptable results, allowing us to focus on systematically
optimising other parameters. Of course, hybridisation
temperature should be empirically optimised for every
probe and has the potential to significantly improve or im-
pair the final result. A summary of the treatments we
found to generate the clearest WMISH signals for each
developmental stage (a “protocol at a glance”) is provided
in Additional file 2. The results of control experiments
using no antibody and no riboprobe (which generated no
signals) are provided in Additional file 3.
The effect of NAC treatment
The fluid that bathes L. stagnalis larvae during their en-
capsulated development is characterised by a high viscos-
ity and adheres to the embryo following de-capsulation.
An incubation step with the mucolytic reagent NAC ap-
parently leads to a superior preservation of the overall
morphology (Figure 2 and Additional file 3C cf. E and 3I
cf. K). However, treatment with NAC resulted in a signifi-
cant reduction of signal intensity for all ages and genes
that we investigated (Figure 2C, G, K, O and S). However,
when NAC treatment was combined with a reduction step
this effect was reversed for some combinations of ribop-
robe and developmental stage (Figure 2H, P and T). The
combined NAC and reduction treatment gave the best sig-
nal to noise ratio for beta tubulin in three to six dpfc lar-
vae (Figure 2H), and for COE in two to three dpfc old
larvae (Figure 2T). This is in contrast to the situation forengrailed in all investigated ages: under the appropriate re-
duction treatment, omitting the NAC generated a better
signal to noise ratio (Figure 2J and N) than including it
(Figure 2K, L, O and P).
Our overall impression of treating L. stagnalis larvae
with NAC is that this treatment may be beneficial in com-
bination with a reduction step when working with probes,
which tend to generate non-specific background. The sig-
nal diminishing effect of NAC is in contrast to the situation
in the planarian Schmidtea mediterranea. Here, a NAC
treatment is used to remove the planarian’s surrounding
mucous layer and generally increases the WMISH staining
intensity, at least when combined with a permeabilisation
step using SDS or DTT [17]. In L. stagnalis, NAC may be
removing the intra-capsular fluid, however it appears
that in our hands NAC is most likely reducing WMISH
signal strength by significantly inhibiting the activity of
Pro-K; larvae that were incubated in Pro-K and 1%
NAC at the same time did not show any signs of com-
promised morphology, while larvae in control reactions
with Pro-K but with less (0.1%) or without NAC were
digested (Additional file 4).
Treatment with DTT and detergents (reduction)
A treatment using a solution containing DTT and the de-
tergents SDS and NP-40 following fixation greatly in-
creased WMISH signal intensity for all investigated genes
and developmental stages (Figure 2). The best WMISH
signal for beta tubulin in four to six dpfc old larvae was
achieved using a combination of NAC and reduction
(Figure 2H). This suggests that the reduction treatment
might represent a highly effective permeabilisation ap-
proach. However, this was at the cost of all material be-
coming highly fragile until dehydrated in ethanol. Reduced
samples were also more likely to reveal unspecific back-
ground staining (Figure 2F, N and R).
SDS treatment
Between one and five dpfc old embryos and larvae of L.
stagnalis were treated with different amounts of the an-
ionic detergent SDS prior to hybridisation (Figure 3). A
permeabilising treatment with 0.1% SDS did not produce
strong WMISH signals for all studied genes and larval
ages (Figure 3A, D, G, J, M, P and S) whereas treatments
with higher concentrations of SDS generated strong
WMISH signals. For two of the genes we studied here,
beta tubulin and engrailed, treating larvae between three
to five dpfc with 0.5% or 1% SDS produced equally good
results. In contrast, the staining was more intense after
treatment with 0.5% SDS than with 1% SDS for COE
(Figure 3Q vs. R and T vs. U) as well as for beta tubulin
in two dpfc old larvae (Figure 3B vs. C), which may sug-
gest a loss of the target transcripts due to excess perme-
abilisation of these younger stages. Additionally, embryos
en
gr
ai
le
d
be
ta
 tu
bu
lin
3 
- 
6 
dp
fc
30
 µ
g/
m
l P
ro
-K
 
2 
- 
3 
dp
fc
20
 µ
g/
m
l P
ro
-K
  
2 
- 
3 
dp
fc
20
 µ
g/
m
l P
ro
-K
 
3 
- 
6 
dp
fc
20
 µ
g/
m
l P
ro
-K
  
2 
- 
3 
dp
fc
20
 µ
g/
m
l P
ro
-K
  
C
O
E
A B C D
E F G H
I J K L
M N O P
Q R S T
+ reduction + NAC + NAC
+ reductionJackson et al., 2007
Figure 2 Overview of WMISH signals generated after pre-hybridisation treatment with NAC and/or reduction. L. stagnalis larvae of
different ages were subjected to a WMISH protocol similar to that described by Jackson et al. [22] (A, E, I, M and Q). This protocol was then
modified by the addition of a reduction treatment (B, F, J, N and R), a NAC treatment (C, G, K, O and S) or a combination of both NAC and
reduction treatment (D, H, L, P and T). Using this set of pre-hybridisation treatments, the optimal sample preparation regime for WMISH varies
with respect to the target gene and the developmental stage. For engrailed and beta tubulin in younger larvae, the samples that underwent a
reduction treatment display the best signal to noise ratio (B, J and N). Excess background that is revealed by reduced samples for COE and beta
tubulin in older larvae (F and R) is diminished by a treatment with NAC (H and T). Black stars indicate optimal results after sample preparation
involving NAC treatment and reduction. Panels A to H show larvae from a lateral perspective with the shell field oriented to the right. Larvae in I
to P are viewed from dorsal and Q to T are viewed from apical.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 7 of 13between one and two dpfc tend to adhere to plastic sur-
faces in 1% SDS. While in other animal systems SDS is
commonly used at a concentration of 1% [18-20], in L.
stagnalis strong WMISH signals were most consistently
achieved with 0.5% SDS across different genes and onto-
genetic stages.
The WMISH signals for engrailed, COE and beta
tubulin (the latter at least in younger larvae) in SDS-
treated larvae reveal equally good or superior signal in-
tensities compared to reduced or NAC-treated and re-
duced larvae (Figure 2 cf. Figure 3). The engrailed signalin the shell field of SDS-treated larvae also clearly exhibits
a significantly improved spatial resolution compared to
the signal in reduced larvae (Figure 2J cf. Figure 3K). In
terms of ease of handling, non-specific background and
consistency of WMISH signal among the different genes,
the SDS treatment is our recommended sample prepar-
ation strategy. A reduction treatment might increase the
signal intensity in WMISH experiments against genes
expressed in older larvae (Figure 2H), but should then be
performed in parallel to the SDS treatment to control for
a possible loss of spatial resolution.
en
g
ra
ile
d
4 
- 
5 
dp
fc
20
 µ
g/
m
l P
ro
-K
 
3 
dp
fc
20
 µ
g/
m
l P
ro
-K
  
2 
 d
pf
c
15
 µ
g/
m
l P
ro
-K
  
C
O
E
0.5% SDS 1% SDS0.1% SDS
b
e
ta
 t
u
b
u
li
n
2 
 d
pf
c
15
 µ
g/
m
l P
ro
-K
 
3 
dp
fc
15
 µ
g/
m
l P
ro
-K
  
4 
- 
5 
dp
fc
30
 µ
g/
m
l P
ro
-K
  
Overview of 
0.5% SDS 
3
 d
p
fc
1
5
 µ
g
/m
l P
ro
-K
  
A B C B’
D E F E’
G H I H’
J K L K’
M N O N’
P Q R Q’
S T U T’
Figure 3 A pre-hybridisation treatment with different SDS concentrations significantly affects the WMISH signal. L. stagnalis larvae of
different ages were subjected to pre-hybridisation treatments with varying amounts of SDS and then hybridised with anti-sense probes to beta
tubulin (A-I), engrailed (J-O) and COE (P-U). For all genes and larval ages, treatment with 0.1% SDS did not generate consistent or strong WMISH
signals (A, D, G, J, M, P and S). Treatments with both 0.5% and 1% SDS produced strong WMISH signals for beta tubulin and engrailed in larvae
aged three to five days post first cleavage (dpfc), with high spatial resolution (inlet in K). For COE 0.5% SDS outperformed the 1% SDS treatment
(T vs. U). Black stars indicate optimal treatments. Note that some treatments produced equally good results. The most consistent results (defined
as constantly good signals among genes and ontogenetic stages with little variation between individuals within an experiment) were achieved
with 0.5% SDS (examples shown in B’, E’, H’, K’, N’, Q’ and T’). Larvae in A-C and M-R are shown from an apical perspective, larvae in D-F are
viewed ventrally, G-I laterally and J-L and S-U dorsally.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 8 of 13
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 9 of 13Enzymatic permeabilisation with Pro-K
The optimal Pro-K concentration for tissue permeabilisa-
tion and mRNA target unmasking depends on the incuba-
tion temperature, incubation time and the ontogenetic
stage of the material. In general, under-treatment yields
weak WMISH signals while over-treatment increases back-
ground staining and leads to a compromised tissue morph-
ology. We tested a wide range of Pro-K concentrations in
combination with different pre-hybridisation treatments
and found that treatment with Pro-K at concentrations of
0–2 μg/ml drastically reduced signal intensity while treat-
ment at concentrations of 40–50 μg/ml compromised tis-
sue integrity (data not shown). Therefore, we finally used
concentrations of 1–15 μg/ml for one to two dpfc old lar-
vae, 5–20 μg/ml for two to three dpfc old larvae and 5–
40 μg/ml for larvae between three and five dpfc in combin-
ation with different SDS concentrations (data not shown).A
E F G
B C
I J K
+ RNAse treatment 
beta tubulin
beta tubulin
beta tubulin
contig 380566
contig 380566
contig 380566
- RNAse treatment /
- RNAse treatment 
positive control
Figure 4 Non-specific probe binding to the shell field and radular is e
and consistent WMISH stain for a variety of probes (represented here with
field (arrows in B and C) and in the radular sac (arrow in D). Probes agains
(A). The stain in the shell field periphery and the radular remains following
signal against beta tubulin is abolished (E). This indicates that the signals in
binding. Treatment with TEAAA abolishes this non-specific stain (J-L), while
J are dorsal views. A, C, E, G, I and K are lateral views of larvae with the sh
G and K are reflected about the vertical axis for consistency of presentationFor embryos between one and two dpfc we found the high-
est concentration of Pro-K of 15 μg/ml gave the strongest
signals (Figure 3B and Q). For two and three dpfc larvae,
15 μg/ml or 20 μg/ml Pro-K were suitable (Figure 3E, F, K,
L and T). In larvae between three and five dpfc the best
signal to noise ratio was achieved with 30 μg/ml for beta
tubulin (Figure 3H and I) and 20 μg/ml for engrailed
(Figure 3N and O). COE is apparently not expressed in lar-
vae between three and five dpfc.
Removal of non-specific background with triethanolamine +
acetic anhydride (TEAAA)
In preliminary experiments using a wide variety of ribo-
probes to different genes we obtained a strong, well-
defined WMISH signal located at the periphery of the
shell field and in the radular sac (Figure 4, black arrows).
To determine whether these staining patterns representedH
D
L
/ - TEAAA treatment
contig 380566 contig 380566
contig 380566contig 380566
contig 380566 contig 380566
 + TEAAA treatment
/ - TEAAA treatment
liminated by treatment with TEAAA. We observe a well-defined
a probe against the gene “contig 380566”) in the periphery of the shell
t other genes (for example beta tubulin) do not produce these patterns
a pre-hybridisation treatment with RNAse (F-H), while the specific
the shell field and the radular are the result of non-specific probe
the specific signal against beta tubulin remains unaltered (I). B, F and
ell gland oriented to the right. D, H and L are ventral views. Panels C,
.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 10 of 13genuine probe/target hybridisation events, or non-specific
binding of the probe, we treated fixed larval material with
RNAse A prior to hybridisation. As a control, all expected
beta tubulin staining was abolished following this RNAse
treatment, confirming the degradation of all target mRNA
(Figure 4E). In RNAse treated embryos hybridised with
probes that generated the spurious shell field and radula
patterns this signal was still present (Figure 4F-H), indicat-
ing these WMISH patterns represent high affinity binding
of the riboprobe to molecules other than RNA. In order
to address this background staining, we assessed the effect
of a triethanolamine + acetic anhydride (TEAAA) treat-
ment. Treatment of biological substrates with TEAAA is a
common practice for many WMISH protocols, and de-
creases non-specific binding of labelled probes through the
acetylation of polar and charged groups [23]. For L. stagna-
lis, treatment with TEAAA following Pro-K digestion+ anti-DIG AP 1:3000
+
optimised
protocol
+ anti-DIG AP 1:3000
+
b
et
a 
tu
b
u
lin
en
g
ra
ile
d
optimised
protocol
A B C
F G H
K L M
P Q R
Figure 5 Our optimised WMISH protocol is not improved by more an
post first cleavage (dpfc) were subjected to our optimised WMISH protoco
of increasing the amount of anti-DIG antibody (B, G, L and Q), the additio
combination of more antibody and PVA (D and I). We also assessed the ef
in combination with a higher antibody concentration and the use of PVA (
results to our baseline protocol. Samples incubated in more antibody and
signal to noise ratio (B, C, G, H, L, M, Q and R). PVA also appeared to com
Signals generated by the hydrolysed engrailed probe were much fainter an
(N, O, S and T). The optimal treatment (A, F, K, and P) is indicated by a b
the increased antibody concentration do not reveal any staining (E and J).successfully abolished the non-specific WMISH signal in
the larval shell and in the radular sac (Figure 4J-L). Due to
its strength, consistency and spatial definition, this tissue-
specific background stain is particularly likely to be misin-
terpreted as genuine WMISH signal. A good example of
this is engrailed, which is genuinely expressed in the shell
field periphery (Figure 3J-O) and also produces the shell
field background stain (data not shown). Our riboprobe
against engrailed (synthesised multiple times) covers the
same region as the probe used in a recent study that pos-
sibly produced the same background staining [14]. There-
fore, treatment with TEAAA appears to be critical for the
correct interpretation of genes with expression patterns as-
sociated with the shell gland and shell field.
General background staining and its elimination by
TEAAA can also be observed for samples that under-
went a NAC (Figure 2) or SDS treatment (Figure 3). For PVA
no probe control for 
anti-DIG AP 1:3000
 PVA
+ PVA
+ anti-DIG AP 1:3000
+ hydrolysed 
  riboprobe 
+ PVA + hydrolysis
+ anti-DIG AP 1:3000
D
I
N
S
O
T
J
E
tibody, PVA or hydrolysed riboprobes. Larvae three to four days
l (A, F, K and P). Using a beta tubulin probe, we investigated the effect
n of PVA to the colour detection solution (C, H, M and R) and the
fect of hydrolysing the engrailed riboprobe individually (N and S) and
O and T). None of these modifications generated superior WMISH
developed with PVA showed slightly more intense signals, but a lower
promise the morphological integrity of older larvae (H, I and R).
d were partially masked by an increase in general background staining
lack star. Control WMISH experiments lacking a riboprobe and using
All images of individual larvae are lateral views.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 11 of 13some genes, the TEAAA treatment can be shortened by
one incubation step in TEAAA instead of two incuba-
tion steps (Additional file 5).
The effect of antibody concentration, PVA and riboprobe
hydrolysis
Since the dilution of AP-conjugated anti-DIG antibody in
our optimised protocol (1/10,000) is lower than described
in many WMISH protocols, we assessed the effect of in-
creasing the concentration of antibody to 1/3,000. While
the signal intensities of beta tubulin and engrailed expres-
sion were slightly higher, more overall non-specific back-
ground staining was also evident (Figure 5B, G, L and Q).
A common approach to improve WMISH sensitivity
and reduce background staining is through the addition
of PVA to the colour detection solution. This is thought
to increase the local concentration of the colour reaction
product by limiting its diffusion [24]. A direct compari-
son of WMISH colour development in L. stagnalis with
and without PVA did not reveal a significant increase in
the signal intensities, but rather a lower signal to noise
ratio for both beta tubulin and engrailed (Figure 5C, H,
M and R). Furthermore, the morphological integrity of
especially older larvae was compromised (Figure 5H).
An alternative strategy to increase WMISH signal sen-
sitivity is to hydrolyse the riboprobe before use [23,24].
Hydrolysing the riboprobe into smaller fragments is
thought to facilitate better tissue penetration and to im-
prove hybridisation kinetics (reviewed in [23,25]). We
specifically tested whether a hydrolysed riboprobe gener-
ates an improved engrailed signal and found that instead
both the signal specificity (as revealed by the lack of theA CB engrailedCOE
E GF
Figure 6 Our WMISH protocol is suitable for fluorescent signal detect
D) in larvae treated with 0.5% SDS was detected using the fluorescent sub
62 hours post first cleavage (hpfc) old larva with the approximate localisati
perspective of a 90 hpfc old larva with indicated engrailed expression. Pane
old larva (G, ventral perspective) and a 100 hpfc old larva (H, lateral perspe
micrographs. Panels B, D and H are reflected about the vertical axis for conshell field lining expression pattern, Figure 5S) and over-
all intensity were reduced compared to non-hydrolysed
control reactions (Figure 5P vs. S). Reduced signal sensi-
tivity derived from hydrolysed riboprobes has been pre-
viously reported [26], highlighting the necessity to test
the optimal probe preparation strategy for each gene in-
dividually [27].
To summarise these treatments, neither more anti-
body nor the use of PVA or hydrolysed riboprobes gen-
erated WMISH signals with higher sensitivity and/or
signal to noise ratios.Fluorescent WMISH signal detection
The optimised protocol that we have identified also al-
lows for the visualisation of fluorescent signals using a
confocal microscope. This method of detection provides
a much higher spatial resolution than colorimetric
methods. Using probes against all three of the genes
employed in this study we were able to develop fluores-
cent signals using Fast Red (Figure 6).The effect of storing fixed material in Ethanol vs.
Methanol
Methanol (MeOH) is used to dehydrate and store fixed
embryonic and larval material at low temperatures (−20°C)
in many WMISH protocols. Due to the high toxicity of
MeOH relative to EtOH we assessed the effect of storing
fixed L. stagnalis larvae in MeOH vs. EtOH on the
WMISH signal generated by beta tubulin, engrailed and
COE. We found no consistent or significant difference with
respect to any of the signals generated (data not shown).Dbeta tubulin beta tubulin
H
ion. The expression of COE (A), engrailed (B) and beta tubulin (C and
strate Fast Red (A-D). Panel E is a scanning electron micrograph of a
on of the COE expression indicated in red. Panel F is a lateral
ls G and H show the positions of beta tubulin expression in a 57 hpfc
ctive). Panels B-D are projections of confocal laser scanning
sistency of presentation. All scale bars are 50 μm.
Hohagen et al. BMC Developmental Biology  (2015) 15:19 Page 12 of 13Conclusions
Our study provides an optimised whole mount in situ
hybridisation protocol for early larval stages of the mol-
lusc L. stagnalis. Using a variety of pre-hybridisation
treatments we have identified a set of conditions that
allow for high WMISH signal intensity and consistency
in colorimetric as well as fluorescent WMISH. These in-
clude a treatment with 0.5% SDS, treatment of one to
two dpfc larvae with 15 μg/ml Pro-K, two to three dpfc
larvae with 15–20 μg/ml Pro-K and three to five dpfc
larvae with 20–30 μg/ml, followed by treatment with
TEAAA. We also demonstrate that non-specific shell
field and radula staining can easily be abolished with this
TEAAA treatment. In our experience, every riboprobe/
developmental stage combination benefits from an indi-
vidualised protocol, which needs to be empirically deter-
mined. Nonetheless we believe that this WMISH
protocol should serve as a baseline method from which
consistent and clearly visible patterns of gene expression
can be obtained. This method should serve to raise the
profile of L. stagnalis as a tractable experimental mollus-
can model, a niche that is currently underpopulated.Additional files
Additional file 1: Primer sequences used to isolate gene fragments
for riboprobe syntheses.
Additional file 2: WMISH “protocol at a glance”.
Additional file 3: Control experiments for the optimised sample
preparations. Control WMISH experiments lacking riboprobe (A-F) or
antibody (G-N) demonstrate the absence of any non-specific colour reaction
for samples treated with SDS (A, B, G, H, M and N) or with reduction solution
(C, D, I and J) as well as reduced + NAC treated samples (E, F, K and L) for
about three dpfc old larvae (A, C, E, G, I, K and M) and about five dpfc old
larvae (B, D, F, H, J, L and N). Panels A to L were colour-developed using
NBT/BCIP as colour substrate and panels M and N were developed using
Fast Red. All embryos are shown from a lateral perspective. Scale bars are
50 μm (A, C, E, G, I, K and M) or 100 μm (B, D, F H, J, L and N). Panels C, E, G
and H are reflected about the vertical axis for consistency of presentation.
Additional file 4: Proteinase-K activity is inhibited by NAC. Larvae
incubated in Pro-K without NAC (A-D) or in 0.1% NAC (E-H) are
almost completely digested after 4 and 22 hours of incubation
respectively. In contrast, larvae incubated in Pro-K with 1% NAC (I-L) do
not show any signs of Pro-K digestion and maintain their morphology
even over extended incubation times (L). All larvae are about 4 days post
first cleavage old. All images are to the same scale shown in L (1 mm).
Additional file 5: A shortened treatment with TEAAA is sufficient to
minimise non-specific probe binding in SDS-treated samples. The
background stain in the shell field periphery (identified in Figure 3) is
also observed for SDS-treated samples (arrows in A), as represented by
a probe against the gene “contig 380566”. Note that after treatment
with SDS, the protonephridia are stained (arrow in B). Both non-specific
WMISH stains are strongly reduced after one incubation step in TEAAA
(D-F) and disappear after two incubation steps (G-I). Larvae in A, D and G
are shown from a dorsal perspective, and larvae in B, E and H are viewed
from lateral. Panels B, E and H are reflected about the vertical axis for clarity
of presentation.Competing interests
The authors declare that they have no competing interests.Authors’ contributions
JH carried out the experimental procedures. IH assisted with the experimental
procedures. DJJ conceived the study. JH and DJJ drafted the manuscript. All
authors participated in the design of the study. All authors read and approved
the final manuscript.Acknowledgements
We gratefully acknowledge Luciana Macis for the maintenance of snail lines
and Diana Bauermeister for isolating engrailed and beta tubulin sequences
used for riboprobe synthesis. We also acknowledge two anonymous
reviewers for providing helpful comments. This work was supported by
Deutsche Forschungsgemeinschaft (DFG) funding to DJJ through the CRC
Geobiology and the German Excellence Initiative, and DFG project number
JA 2108/1-1.
Received: 15 December 2014 Accepted: 4 March 2015
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