[Histonet] (no subject)

[Galina Deyneko]

Hi Marcel, 
It looks like you wash out cells on previous steps, pipette the reagents into well very slowly, do not pipette on the membrane directly, only underneath of membrane into well using small holes.
Do not use tissue processor, process only manually.
I believe you use xylene substitute. I use Shandon HistoSolve # 9990505 from Thermo Shandon.
Last step in processing is paraffin.
Use intermediate step 1 part of xylene sub+ 1 part of liquid paraffin.
In the oven 58- 60 C i have 3 containers
1:1 Xylene sub: paraffin- 30 min
Paraffin 1- 1 hour
Paraffin 2- 1 hour.
I take out insert (not membrane) from well  and incubate in consecutive order in containers.After last paraffin I take out insert,  carefully poor off the excess of paraffin and leave thin layer of paraffin on both side of membrane and let solidify at room T. Layers prevent cells on membrane. Using warm surgical blade cut out membrane covered by paraffin from insert, cut on 2  or 3 pieces by scissors and embed on routine way, it means you embed a piece of paraffin in paraffin.
Galina.



		
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