Hy,

mix 126 ml Glycerol (87 %), 150 ml Ethyleneglycol and 250 ml Phosphate buffered saline (PBS, 0,1 M, pH 7,4).

For rat brains (I use exactly the same pretreatment procedure you described above) I then take 48wells (old wells from cell culture that I cleaned) and fill each well with 0.5 ml of this solution (Mouse: 96 wells and 0.2 ml) .You can then immediately transfer your brain slices into this solution (at room temperature) and afterwards you cutted the whole brain (or your region of interest) you can wrap them in aluminium foil, label them and store this wells at -20°C for several years and then perform immunohistochemistry. You have to take care that in the freezer they stand every time in a horizontal manner.

Before you start your stainings wash the slices 3 times for 10 min in PBS.

By using different wells for one brain you can immediately generate different series for different stainings and then collect series from various animals. This allows you to stain the same serie from different animals with the same primary antibody and make stainings comparable.

Hope I could help you.

Stefan

Dear Stefan Jean-Pierre Haas,

Your suggestions would be a great help for me. Thank you

Question is not clear: if you want to store before cryosectioning use the fixative and store at 4 Celsius in the frigidair. After sectioning we used the plastic 12 egg holders from the shop, filled with the perfusion fixative and stored at 4 Celsius in the frigidair. Before staining rinse with PBS.   

Dear Enrico Marani Sir,

Thank you for your suggestion. I would like to preserve the brain tissue before  performing the cryosectioning 

Dear Mohamed Mahdy,

Thank you for your suggestion. 

Go to chapter 12 of Ultramicrotomy by Norma Reid in the series Practical methods in electron microscopy by series ed A.M.Glauert. Noth Holland /American Elsevier 1975. Rather old, but in that time EM made its future.. Good luck Marani