Hi Paula:

Excise the tissue. Fix tissue in 3 mL-5ml of freshly diluted cold 4% PFA, keep on ice for 1 h, wash with cold PBS three times for 10 min each, transfer to cold 15% cold sucrose solution (made in PBS) with gentle rocking for 3 h (in cold room or refrigerator at 4C), and finally transfer to 30% cold sucrose and keep overnight in a cold room at 4/5° with gentle rocking.

Rinse the tissue thrice, 5 min each in OCT (Tissue-Tek®), and place in a Cryomold® of the appropriate size (Tissue-Tek) filled with OCT. Freeze the blocks at -70 or on dry ice. Semi-thin sections of 7 μm thickness can be cut using a Cryostat. Store frozen section slides in a slide box at -70 or 80C.

View sections in light microscope

If you want to orient the tissue: place the mold on dry ice, add few drops of OCT, place tissue and quickly orient the tissue with a fine pair of foreceps, add more OCT to fill the mold and use the frozen block as said before.

You may completely avoid Sucrose step if you like,

If you have any other concern, please let me know.

Good luck

Hi Paula,

PFA does quench GFP, but there are good polyclonal anti-GFP antibodies you can use for IHC, which still recognize fixed GFP. We usually go in afterwards with a secondary coupled to AF488. The anti-GFP living colors from Takara is good (polyclonal rabbit) but gives in my hand a little background) and there is also a very good polyclonal IgY anti chicken sold by a couple of different companies (e.g. here: https://www.thermofisher.com/antibody/product/GFP-Antibody-Polyclonal/600-901-215). But then you need an anti IgY-AF488 which is maybe a bit exotic. But a good idea because using IgY/anti-IgY is usually very clean in mammals.

You can use antibodies that target directly against GFP as an alternative, if you are worried about fluorescence quenching due to PFA.