Dear fellow knowledge seekers.
I have a possibly quick question to ask to the IHC experts in here.
Following scenario, I have a couple of unperfused snap frozen brain tumors and I'd like to make slices on a cryostat from them and subsequently use them for IHC.
The question is, do I either cut the tissue directly at the cryostat and PFA fix the slices afterwards or do I thaw the sample and fix the tissue beforehand? The issue with the 2nd option could be that the tumor samples have a diameter of a fingernail so the PFA might not be able to penetrate the sample well enough then?
Anyways, I am grateful for any tips or hints you can give.
Cheers
Niklas
It depends on your antibodies you like to use, because not every antibody works on native, unfixied tissue. Take one tissue sample without fixation (native) and one sample which you fix in PFA. After fixation you have to cryoprotect the tissue in 30% sucrose (until it drops on the bottom of the vial) before cutting it.
Take 10µm thick sections from the native tissue. Mount them directly on the superfrost plus slides, air dry them for 30 min an put them into a slide box which you store at -80°C until you will use the section for your immuno. Before you start the IHC put the sections overnight into 4% PFA at 4°C after 1 hour equilibration to room temperature. Next morning short rinse in destilled water; 3x5 min PBS; 2 rinses in 70% Ethanol (ETOH), 100% ETOH 20 min at 4°C (if you use peroxidase labeled secondary antibodies or detection systems like streptavidin deactivate the endogeneous peroxidase with H2O2; H2O2 1:100 in 100%ETOH for 20 min at 4°C); 2 rinses in 70% ETOH, 3x5 min PBS; continue with your IHC protocol.
The sections of the fixed tissue mount in the same way you did with the unfixed ones. After air drying you can store them at -20°C until you will start with your IHC.
After equilibration at room temperature you can start with 3x5 min rinsing in PBS and the IHC protocol without additional fixation step. Compare the result of both kinds of tissue preparation and the best result use for the other semples. Good luck!
Thank you for your question Niklas.
You can check these sites may help.
All the best.
https://www.biolegend.com/en-us/protocols/immunohistochemistry-protocol-for-frozen-sections
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections
PFA should be able to fix the tissue. you could also fix before you cryosection that way you could do both cryosectioning and IHC. It is also possible that you may be try dropping the cry tissue block in PFA so that as it melts, it is already coming into contact with the PFA. Ia m not sure if things like OTC will dissolve in PFA like it does in water. I would then transfer the tissue to fresh fixative once it has thawed and then proceed with the IHC.