Dear Davis,

I am not sure I am understanding your question correctly.  Do you mean that you want to immersion fix the frozen tissues and then section them?  I have not tried that, since I prefer to either perfuse my animals and cryoprotect the tissue samples, or freeze the tissue without fixation, before I cut sections.  I would think that the thawing that occurs while the tissue is immersed in fixative would be bad for the integrity of the whole tissue, not just for the antigen.

I have fixed thin (10 um) frozen sections that were cut from unfixed material with 4% paraformaldehyde on the slide after sectioning and have been able to perform immunohistochemistry, but the success will depend on your antigen.  A long time ago there was a neighboring lab that had to fix their unfixed frozen sections with paraformaldehyde vapor (in a well sealed glass dish in the fume hood) because they couldn't put the sections into the liquid phase or they would lose a critical component.

Good Luck,

Jill

Thanks, Jill. You understood the question correctly. We've successfully used this process to make H&Es from small frozen specimens without any noticeable effect on morphology (although we haven't tried IHC with these).

It sounds like fixing the frozen sections with paraformaldehyde might be a viable option. In the cases where this did work, did you include an antigen retrieval step?

Thanks again,

Davis

We have done this with no problem -fix in formalin - process as normal. IHC should work with the same protocol used for FFPE tissue.

Dr. Amary,

It's good to hear someone has had success with this. We'll give it a shot.

Thanks,

Davis

Hi Davis,

I am glad someone answered who has done what you wanted.  I agree, if they are very small bits of tissue, it will probably work with formalin.  Since I am a neurobiologist I freeze whole rodent brain with powdered dry ice, and I would not expect it to fix well during while thawing.  Also, I use paraformaldehyde in order to avoid the methanol in formalin.  When I use 4% paraformaldehyde after frozen sectioning, I do not use an antigen retrieval method.  However, the antigens of interest for me do not usually need a retrieval method.

Here's hoping that your new method is successful.

Best,

Jill 

Hello Davis, 

Yes you can fix your frozen tissue in 10% formalin, embed in paraffin wax and perform your IHC on it. I however think that you have to perform antigen retrieval prio to the IHC.

A quick question to you, Jill, may i know which antibodies you usually work with?

Dear Bernice,

I use 10-12 um cryostat sections of paraformaldehyde-fixed material (not paraffin sections), and usually work with antibodies to cytoskeletal elements present in neurons and glia (anti-GFAP, all 3 weights of neurofilament, different anti-tubulin Abs, anti-Tau, etc).  These proteins are usually robust and abundant, so no antigen retrieval is necessary.  My publications also include work with anti-GAP 43 and anti-CNTF receptor alpha subunit.  These antigens are a little more sensitive to poor handling because of their linkage to the cell membrane, but again, I did not find antigen retrieval necessary.  The only frozen material that I fixed on the slide post sectioning was used to detect vimenten and GFAP in rat brain.  Again, these are abundant antigens, so I didn't have a problem.  When I have suspected a problem I have used a glycine or lysine rinse to quench any excess paraformaldehyde, but I usually only do this for tract tracing (anti-Cholera toxin B fragment), or when I use a fluorophore that emits a green signal.

What I have found to be more critical in terms of detecting an epitope is 1) section storage (not for long, and don't let them dry out in the freezer), and 2) unnecessary drying of sections.  I just let my sections attach in the cryostat by storing the slides in a slidebox in the cryostat chamber while I section; I do not air dry them out on the bench.  In my hands that leads to unacceptable levels of background staining, as well as loss of appropriate signal.  Since I section posterior eye cup and retina with the optic nerve attached, I want to make sure that the material stays attached to the slide.  I do this by holding my finger under the slide in the area of the section so that the section melts onto the slide very well, but I do this with my hands in the cryostat chamber.  Needless to say, my hands get very cold while I section, and I wear fingerless cotton gloves to help protect them. 

To make sure the sections do not detach due to cryocondensation of water under the material, I always let my (well-sealed) storage boxes come to room temperature after they are removed from the -80 C before starting the immunohistochemistry protocol.

If you need any further details, please let me know.

Best,

Jill

Hi Davis R Ingram,

I was wondering if you were successful with your attempt to do IHC in fixing your frozen tissues in formalin for IHC?

I am planning to do the same in brain tissue samples.

Thank you.