IHC staining protocol for whole mount samples

Protocol and troubleshooting tips for whole mount immunohistochemical staining.

Whole mount staining is the staining of small pieces of tissue – usually embryos – without sectioning. 

Whole mount staining is very similar to immunocytochemistry (ICC) or staining of cryosections. If an antibody has been used successfully on cryosections (this does not include paraffin-embedded sections), then the antibody should work for a whole mount embryo.

The difference is 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 center of the sample. 

The timing of these steps will need to be optimized for your experiments, but the details in these protocols provide a guideline.

Whole mount fluorescent immunohistochemistry protocol

Chick or mouse whole mount immunohistochemistry protocol

Drosophila whole mount immunohistochemistry protocol

Whole mount troubleshooting tips

Fixation

Whatever fixative you have successfully used in IHC-Fr with your antibody should also 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 permeabilization to the center 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 cross-linking, 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 ensure the egg membrane is permeabilized.

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 petri dish, before mounting. If small enough, the whole embryo can be mounted in glycerol before setting in a cover slip. In this case, grease should be used around the corner of the cover slip to help keep it in place.

Embryos can also be set in gelatin 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.

Choosing the age of the embryo

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 center 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