[Histonet] Immunofluorescence

[Daniela Bodemer]

Hi Natalia,

Human tissue is naturally autofluorescent. In our lab we do immunos on
frozens and quench the AF with chicago blue solution (1g Chicago Sky
Blue 6B-Sigma in 99ml MilliQ plus 1ml DMSO) after washing the secondary
antibodies with PBS (3x). Just load the tissue with a drop of the CB
solution for 15 secs and wash it in PBS again (3x). Mount as usual

D

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Natalia
Fernandez
Sent: Wednesday, 8 February 2012 2:35 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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