I rely on numerous articles, however, in regard to the removal of the historesin, I face some difficulty. When we talk about removal of historesin, would this resin be completely removed? Or is the removal only intended to form pores in the sections?
1 bath 2methoxyethyl acetate 20min
1 bath 2methoxyethyl acetate 15min
ethanol 70% 10min
ethanol 40% 10min
tap water 5min
My eaching protocol:
semithin sections should be dried out on the slide;
10 % Na-Methylate in Methanol -- 1h
10 % Na-Methylate in Methanol/Aceton à/à -- 30 min
Aceton -- 10 dips
Methanol -- 2 x 21/2 min
PBS -- 4 x 5 min
This precess does not only increase the pores of the resin, it eaches the resin away. The other protocols do it in the same way.
Contimue with your immuno protocol. I have worked with this Protocol on 1 µm thick optic nerv sectons. I used GFAP, Vimentin, Myelin Basic Protein and Neurofilament 160 antibodies with good results. Another problem could be the OsO4 pretreatment before embedding. Osmium could cover the epitopes.
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