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

From:"Tan, MinHan"
Message
Sorry, I neglected to mention -
 
I have also tried
 
Sections were cut in cryostat and stored as unfixed slides overnight in -80. (no dessication though)
Unfixed frozen slides (stored at -80) placed at -20 for 5 minutes
Placed in -20 acetone for 5 minutes.
Air dried at room temperature for 10 minutes

As per standard=20 protocol.
 
 
-----Original Message-----
From: Tan, MinHan
Sent: Wednesday, November 19, 2003 5:33 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Frozen section fixing problems - but paraffin works!

Hi,
 
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=20tissue, 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=205% x 30 min, primary antibody (mouse) 4 deg overnight, secondary (goat anti-mouse), ABC, DAB.
 
Does anyone have any advice?
 
Thank you!

Regards,
Min-Han Tan 
 
 
 
 
 
 
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