Everyone ever do the immunohistochemistry without tissue being fixed with formaldehyde? After sacrifice the animal I directly put the tissue into the 30% sucrose, for further cryosection, I totally forgot to do the fixation process. Just want to ask what will happened if I missed the procedure. Thx
No trouble.
To perform an IHC on a frozen section, the sampling block can be frozen without being fixed. The fixation / permeabilization takes place immediately after cutting and depositing on the slide.
I do not use formaldehyde because most antibodies do not work with this type of fixative.
I prefer the fixation / permeabilization with cold acetone because most of the antibodies give a better answer without the tissue being too much altered by this mode of fixation
No trouble.
To perform an IHC on a frozen section, the sampling block can be frozen without being fixed. The fixation / permeabilization takes place immediately after cutting and depositing on the slide.
I do not use formaldehyde because most antibodies do not work with this type of fixative.
I prefer the fixation / permeabilization with cold acetone because most of the antibodies give a better answer without the tissue being too much altered by this mode of fixation
well it depends how long the tissue spent in the sucrose before freezing and on what temperature. Like Jean I prefer not to use PFA, still sometimes i have to even do perfuse fixation.
my suggestion to you is to post fix tissue with either acid alcohol or gluteraldehyde/PFA, and maybe you can still get away with some intact structures.
I am assuming you are using a cryostat because you used sucrose solution.
All the best
Amjad
Either snap freezing, like Christoph has discribed it, or fixaton with formalin (paraformaldehyde) or acolhol/ aceton is nacessary to prevent the tissue from autolytic processes. If you have stored the tissue in 30% sucrose for a longer time before you do the cryosectioning it could be distroyed. To check the quality ot the structure I recommand to do a H&E staining or Nissl-staining (brain tissue) before starting the IHC.
Shock freezing with isopentante chilled in liquid ntirogen is a good way. But be careful. In our lab, the glass lining containing liquid nitrogen, where a small container with isopentane was suspended, explode with glass splinters all over the place. So better wear googles (it always good when you work with liquid nitrogen)
Yu Chieh Ho, Your method is totally fine if your tissue left in sucrose O/N at 4oC. Do fixation after IHC. But I think dry ice chilled isopentante will be better than liquid N2.
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