[Histonet] Immunofluorescence

[Leiker, Merced]

Paraffin does indeed give a lot of autofluorescence. We use a solution of 0.3% Sudan Black in 70% Ethanol for 10 minutes on the tissues to help with that after the staining. It doesn't mask the autofluorescence completely but reduces it enough to give us good contrast of signal against the background. Mostly we look at cardiac tissue. Maybe someone will have a better idea for brain...

Regards,
Merced

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Natalia Fernandez
Sent: Tuesday, February 07, 2012 10:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Immunofluorescence


Hi everybody!
I have a problem with my experiment.
 
I try to do immunofuorescences in old human brains (parafine sections).
First I thought that the too much colocalisation was due to my antibodies (primary or secondary).
After several tests (Only 1st antibody, only 2nd one, simple staining, inverse simple staining,..) I saw that the tissue itself shows a lot of fluorescence (without antibodies, and even after a treatment against lipofuscine (solution of Potassium permanganate).
 
So my question is: Do you have the same problem?
Do you think that parafine could be the reason of this autofluorescence?!
What can I do?
 
Thank you so much for your answers :)
Natalia 		 	   		  _______________________________________________
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