----- Original Message -----

From: Tan, MinHan

To: histonet@lists.utsouthwestern.edu

Sent: Wednesday, November 19, 2003 7:32 PM

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 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!

Regards,

Min-Han Tan 

 

 

 

 

 

 

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