Re: [Histonet] Frozen section fixing problems - but paraffin works!

From:"Hernan Aldana Marcos"

Dear Tan
You have a lot of advaices in the wonderfull manual of Technical Immunohistochemistry of Rodney Miller, go to and download it free.Or go to and donwload the documents named INMUNO1.... to INMUNO5.
Dr. Hernán J. Aldana Marcos
Facultad de Medicina. Universidad de Morón
Machado 914. B1708JPD. Buenos Aires. Argentina
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----- Original Message -----
From: Tan, MinHan
Sent: Wednesday, November 19, 2003 7:32 PM
Subject: [Histonet] Frozen section fixing problems - but paraffin works!

I am working with a new monoclonal antibody in parathyroid tissue, and I use the ABC-DAB reageant system for immunohistochemistry.
I am new to frozen sections immunostaining - and I am encountering problems with frozen sections - staining is very weak or absent, in comparison to paraffin embedded tissue, which I have no problems with. 
My protocol for frozen sections:
Unfixed frozen slides with 5 micron sections thawed at room temperature for 5 minutes
Placed in 70% ethanol x 5 minutes.
Washed in PBS x 5 minutes.
followed with standard protocol: hydrogen peroxide 0.3% x 30 min, donkey serum 5% x 30 min, primary antibody (mouse) 4 deg overnight, secondary (goat anti-mouse), ABC, DAB.
Does anyone have any advice?
Thank you!

Min-Han Tan 

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