Whole Mount Immunohistochemistry Protocol
Provided by:
Professor Anthony Graham Dr. Mattieu Vermeron MSc. Kate Hayes
Kings College London, University of Cambridge, Abcam plc, Cambridge,
UK UK UK
Professor of Developmental Research Fellow Scientific Support
Biology and Molecular Department of Physiology, Specialist,
Neurobiology Development and
Neuroscience
Whole Mount Staining in Chick embryo:
Rabbit anti-MYC (ab9106) and Goat anti-Rabbit FITC Hybridoma bank anti-Islet1 and Goat anti-mouse Alexa 568
www.abcam.com/protocols or technical@abcam.com
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Important notes before starting
1. Note on Fixation:
Whichever fixative has been successfully used in IHC-Fr with the antibody you have chosen should be suitable
for whole mount. However, most researchers use 4% paraformaldehyde (PFA). Although this concentration of
PFA is very low, this has to be left on for a long period of time on whole mount samples to allow for
permeabilization to the centre of the sample. Therefore, this will not be suitable for all antibodies, as the protein
cross linking formed by the fixative may block access of the antibody to the epitope. Normally, in IHC-P, you could
perform antigen retrieval. This is not possible on embryo samples as the heating procedure would destroy the
sample. If PFA fixation does not work for the whole mount tissue, then there is a possibility the antibody is
sensitive to the protein crosslinking, and you will require another fixative. Methanol is a popular second choice of
fixative when optimizing whole mount procedures.
Zebrafish embryo fixation and preparation requires extra steps to fix and permeabilize to ensure the egg
membrane is permeabilized. See Zebrafish whole mount protocol for details.
2. Obtaining images:
Some researchers view and obtain images of embryos as they are. The whole embryo can be imaged while
floating in glycerol buffer in a petridish, before mounting. If small enough, the whole embryo can be mounted in
glycerol before setting in a coverslip. In this case, grease should be used around the corner of the coverslip to
help keep it in place and prevent damage to the coverslip when using the microscope. However, they can also be
set in geletin and sectioned if it is difficult to obtain a clear view of the staining through the whole embryo
(particularly at larger late embryo stages or larger tissue samples).
If immunofluoresent labeling is used, then confocal microscopy can be a useful tool to scan through the embryo,
rather than sectioning the whole embryo onto separate slides after staining.
3. Choosing the age of the embryo:
This is important as the embryo grows, it will become too large to stain. The various reagents, including fixative,
antibody and developing solution will not be able to permeate to the centre of the sample, and the number of
stained cells will make obtaining a clear image very difficult. However, larger and older embryos can be dissected
into segments before staining if necessary.
Recommended ages:
• Chicken embryos: up to 6 days
• Mouse embryos: up to 12 days
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PROTOCOL
Chick or Mouse Whole Mount
Immunohistochemistry
Staining Procedure
1. Obtaining the embryo:
Chick: Gently break the egg into a medium sized clean glass Petri dish. The embryo will naturally float to the top
of the yolk. It will then be visible for careful removal using clean scissors and a Pasteur pipette with the tip
removed (this prevents any damage to the embryo from the narrow end of the pipette).
Mouse: Operate on adult female to remove embryos.
Dissect the embryo in ice cold PBS removing as much unwanted tissue as possible.
We recommend removing as much embryonic membrane and excess tissue as possible as this can
prevent the antibody perfusing into the embryo.
2. Place embryo in a 5 ml bijous in 4% paraformaldehyde. Leave to fix 4°C. The time required will need
optimization. We suggest trying between 2 hours and overnight.
OR Fix in m-DMSO (80% methanol, 20% DMSO).
Generally whichever fixative has been used successfully with the antibody when used in cryosections,
This fixative should be suitable for whole mount. However, this may require some optimization.
When the sample is fully equilibrated with the fixative (i.e the fixative has permeabilized the whole sample) then it
should sink to the bottom of the solution. Ensure the sample has sunk to the bottom of the fixative before
proceeding.
3. Wash 3X in PBS 0.5 - 1% Triton 30 min each time.
4. Incubate the embryos twice for 1 hr in block (PBS 1% Triton + 10% FCS, 0.2% sodium azide), room
temperature.
5. Wash embryos 2X in blocking buffer.
6. Transfer embryos using Pasteur pipette with the end cut off to a 2 ml tube. Add primary antibody at the
required dilution / concentration.
It is recommended that as incubations can be very long in whole mount staining, the antibody should be
diluted in blocking buffer containing 0.02% sodium azide to prevent microbial growth.
7. Incubate for 1 to 4 days on a gentle rotation devise at 4°C.
This incubation time will require some optimization depending on the antibody and also the size of the
embryo.
8. Wash embryo’s 3X 1 hr in PBS 1% Triton + 10% FCS.
9. Wash 3X 10 minutes in PBS 1% Triton.
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10. Wash embryo’s 3X 1 hr in PBS 1% Triton + 10% FCS 0.2% sodium azide.
11. Wash 3X 10 minutes in PBS 1% Triton.
12. Add secondary antibody in blocking buffer (PBS 1% Triton + 10% FCS + 0.2% sodium azide).
13. Incubate for 2 to 4 days with gentle rotation 4°C.
14. Wash 3X 10 minutes in PBS 1% triton.
15. Mount and view embryo’s. Store at 4°C in the dark until analysis.
Mounting Procedure
1. Place sample in 100% glycerol for 48 hours. When sample is fully equilibrated with the glycerol (i.e it is fully
perfused with the glycerol) it will sink to the bottom of the vial. Ensure the sample is at this stage before
proceeding.
2. 75% glycerol has approximately the same density as gelatin which is used to mount and set the samples on a
slide. Therefore, samples should be equilibrated in 75% glycerol after staining for approximately 15 minutes
(again, when equilibrated, the sample should sink).
3. Place in 50% glycerol until the sample sinks. The embryo can be imaged at this stage, or mounted whole in the
glycerol on a slide. Use grease around the edges of the coverslip for protection.
If the sample is to be embedded in gelatin and sectioned on a vibratome; place 20% gelatin pre-warmed to 65oC.
Leave for approximately 30 minutes to equilibrate before taking out the sample to mount. When equilibrated in the
gelatin, the sample should sink to the bottom.
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Protocol Diagram
1. PREPARE EMBRYO
Chick: Careful removal using clean scissors and a Pasteur pipette with the tip removed
Mouse: Operate on adult female to remove embryos.
Dissect the embryo in ice cold PBS removing as much unwanted tissue as possible.
2. FIX
4% paraformaldehyde 4°C. Optimize time between 2 hours and overnight.
OR fix in m-DMSO (80% methanol, 20% DMSO) or other fixative of choice.
3. WASH
3X in PBS 0.5 - 1% Triton 30 min each time.
4. BLOCK
2X 1 hr in block (PBS 1% Triton + 10% FCS + 0.2% Sodium Azide), RT.
5. PEROXIDASE BLOCK
Incubate in peroxidase block (0.1% H2O2 diluted in blocking buffer) overnight 4°C.
6. WASH
2X in blocking buffer
7. PRIMARY ANTIBODY
Add primary antibody at the required dilution / concentration. Incubate 1 to 4 days (will require
optimization) on a gentle rotation devise at 4°C.
8. WASH
3X 1 hr in PBS 1% Triton + 10% FCS 0.2% sodium azide
3X 10 minutes in PBS 1% Triton
9. SECONDARY ANTIBDY
Add secondary antibody in blocking buffer (no sodium azide)
Incubate for 2 to 4 days with gentle rotation 4°C
10. WASH
3X 10 minutes in PBS 1% triton
11. DAB COLOUR DEVELOPMENT
Incubate in DAB substrate for 2 to 3 hrs RT.
Transfer embryos to a dish and add fresh DAB plus 5 μl H2O2 per 1 ml of DAB.
12. STOP COLOUR DEVELOPMENT
Rinse three times in PBS once reaction and staining have reached desired intensity
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Troubleshooting Tips
Very similar difficulties to immunocytochemistry (ICC) and immunhistochemistry (IHC) can occur when staining
whole mount tissue. We can recommend reviewing the ICC and IHC troubleshooting tips provided on the
following page:
http://www.abcam.com/ps/pdf/protocols/abcam%20troubleshooting%20tips%20-%20IHC.pdf
The following tips are more specific to whole mount staining. Most of these relate to the fact that incubation times
for all reagents, and wash steps, need to be much longer that in ICC or IHC to allow penetration through the
sample, which will be much larger than a tissue section.
High background
Fixative used is not suitable for the antibody
Most researchers use PFA for fixation. As antigen retrieval methods are not recommended for whole mount (it
can destroy the tissue), ensure the concentration of this is no more than 4%. This should cause fewer difficulties
with protein crosslinking. Some antibodies will still be sensitive to the small amount of protein crosslinking at this
lower percentage PFA, and PFA fixation will not be suitable for some antibodies.
The usual alternative to PFA is methanol fixation. However, we would recommend checking the antibody
datasheet to obtain information on fixation agents used successfully in whole mount sections with the antibody
you are using.
If this information is not available, fixatives used successfully in cryosections are usually successful in
wholemount.
Fixation time may also require optimization.
Antibody left on for too long
The recommendation when optimizing antibody concentration in whole mount is to start with 3 or 4 day
incubation.
If this is not successful, work backwards to 1 day (antibody will need to be on the sample for at least 24 hours to
ensure full penetration through the sample).
Microbial contamination
As the incubations in whole mount staining are very long, microbial contamination can become a problem. This
can lead to non specific background staining. Use clean glassware (preferably sterile) and fresh reagents. 0.2%
sodium azide can be added to antibody buffers and blocking buffers wherever possible. Please note this should
not be added to peroxidase conjugated secondary antibodies as it can inhibit the enzyme acitivity. Follow washing
guidelines carefully before adding peroxidase conjugated secondary antibody to ensure any sodium azide is
washed away.
Wash steps not sufficient
Follow the wash step guidelines provided in the protocols. Wash steps will need to be long enough to permeate
and wash through the whole sample. Triton rather than Tween is used as this is a stronger detergent which will
permeate more easily.
No signal
Antibody not left on for long enough
Antibody incubations should be much longer than when staining sections on slides to ensure adequate
permeation to the centre of the sample. The recommendation when optimizing antibody concentration in whole
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mount is to start with 3 or 4 day incubation. If this is not successful, work backwards to 1 day (antibody will need
to be on the sample for at least 24 hours to ensure full penetration through the sample).
Incorrect incubation times
Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development
will need to be much longer to allow for permeabilization right into the centre of the sample.
Fixative used is not suitable for the antibody
Most researchers use PFA for fixation. As antigen retrieval methods are not recommended for whole mount (it
can destroy the tissue), ensure the concentration of this is no more than 4%. This should cause fewer difficulties
with protein crosslinking. Some antibodies will still be sensitive to the small amount of protein crosslinking at this
lower percentage PFA, and PFA fixation will not be suitable for some antibodies.
The usual alternative to PFA is methanol fixation. However, we would recommend checking the antibody
datasheet to obtain information on fixation agents used successfully in whole mount sections with the antibody
you are using.
If this information is not available, fixatives used successfully in cryosections are usually successful in whole
mount.
Fixation time may also require optimization.
Incorrect detergent used in buffers
In order to allow full permeabilization of reagents and antibody through the whole sample, Triton rather than
Tween is normally used. This is a stronger detergent which will permeate more easily. It is also used at a
relatively high concentration of 0.5 to 1%.
Zebrafish – egg membrane not permeabilized
Whole mount staining of Zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg
membrane is permeabilised. Fix for 1 hour, wash in PBS 1% triton then permeabilize the egg membrane in in ice
cold acetone / PBS for 8 minutes only. We recommend following the zebra fish whole mount staining procedure
provided.
Mouse and chick – extracellular membrane not removed
Remove as much embryonic membrane and excess tissue as possible as this can prevent the antibody perfusing
into the embryo.
Patchy staining
Incorrect incubation times
Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development
will need to be much longer to allow for permeabilization right into the centre of the sample. If any of these
reagents have not penetrated the whole sample, there may be areas of the tissue not fully washed, fully fixed, or
with full access to the antibody. This will lead to patchy areas where staining is not sufficient.
Air bubbles in the tissue / inadequate mixing of reagent and sample
Ensure the sample is placed on a gentle rotating or rocking devise whilst incubating to prevent formation of air
bubbles and to ensure access of reagent to all the tissue. If any of the reagents have not penetrated the whole
sample, there may be areas of the tissue not fully washed, fully fixed, or with full access to the antibody. This will
lead to patchy areas where staining is not sufficient, or trapped airbubbles in which tissue will not be stained.
Incorrect detergent used in buffers
In order to allow full permeabilization of reagents and antibody through the whole sample, Triton rather than
Tween is normally used. This is a stronger detergent which will permeate more easily. It is also used at a
relatively high concentration of 0.5 to 1%.
Sample is too large.
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An embryo will grow to a stage where they are too large for whole mount staining with good results, and other
types of sample will need to be kept within an optimized size range. Reagents will not be able to fully penetrate
the tissue if it is too large, and the staining through the sample will be patchy. It is sometimes possible to dissect
the sample into sections which are more manageable.
Morphology of the tissue is not good
Inadequate fixation or over fixation
Ensure the sample has been fixed for long enough to allow penetration of fixative through the sample. The timing
of fixation may require some optimization. Fix at 4°C. We suggest optimizing between 2 hours and overnight.
Sample has been heated or treated for antigen retrieval
Heat treating the whole mount samples for antigen retrieval is not possible, as it destroys the structure of the
tissue. Try using another fixative, rather than PFA. The usual alternative to PFA is methanol fixation.
However, we would recommend checking the antibody datasheet to obtain information on fixation agents used
successfully in whole mount sections with the antibody you are using. If this information is not available, fixatives
used successfully in cryosections are usually successful in whole mount.
Sample has been crushed whilst handling
To move the tissue from one vial to another, or to add or remove reagents, use a plastic Pasteur pipette with the
tip removed (this prevents any damage to the embryo from the narrow end of the pipette). Avoid use of forceps
where possible.
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What you need to know about Whole Mount
Immunohistochemistry
Whole mount staining is the staining of small pieces of tissue, usually embryos, without sectioning onto slides
first. This is often used on embryos by stem cell and embryonic development researchers and also
neuroscientists who are able to stain the whole embryos at various stages to follow the expression of target
proteins through the development of the animal.
Whole mount staining is very similar to immunocytochemistry (ICC) or staining of cryosections. If an antibody has
been used successfully on cryosections then the antibody should work for a whole mount embryo. The difference
being that the sample being stained is much larger, and thicker, than a normal section on a slide. Therefore,
incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development
will need to be much longer to allow for permeabilization right into the centre of the sample. Researchers use
different times, but the details in these procedures provide a guideline for optimizing the experiment at these
stages if necessary.
The following protocol is for Chick or Mouse Whole Mount Immunohistochemistry. Related protocols are also
available for:
• Whole mount fluorescent immunohistochemistry protocol
• Chick or mouse whole mount immunohistochemistry protocol
• Drosophila whole mount immunohistochemistry protocol
• Zebrafish whole mount immunohistochemistry protocol
• Whole mount troubleshooting tips
All Abcam protocols are available at www.abcam.com/protocols.
www.abcam.com/protocols
