[Histonet] Immunocytofluorescence on smooth muscle cells

[Katri Tuomala]

Hi Bertrand,
Two things come to mind:

1. Your blocking serum should be from the same species as what your
biotinylated antibody was raised in (ie.goat in your case).

2. Rabbit antibodies sometimes can non- specifically bind to smooth muscle
cells and I don't know if there is a way to prevent it.

Hope this will help a bit.....

Katri

Katri Tuomala
Anatomic Pathology
St Joseph's Health Care
Hamilton, Ontario, Canada
----- Original Message ----- 
From: "Bertrand Lefort" <lefortb <@t> crhsc.umontreal.ca>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Sunday, September 29, 2002 10:01 AM
Subject: [Histonet] Immunocytofluorescence on smooth muscle cells


> Hello,
>
>
> We are a new lab and we try to develop Immuno-cyto-fluorescence techniques
> in the lab.
> We are working with human bronchial smooth muscle cells.
> I have currently a very big problem with all rabbit antibodies. All rabbit
> antibodies (including IgG as isotype) give a non-specific signal, signal
in
> the nucleus and cytoplasm with very high intensity. There is no signal
> between cells.
>
> This problem does not exist with Rat and mouse antibodies I have used.
>
> - I have tried different fixation methods (PFA 4%, acetone-methanol (1/1),
> and kit like permeafix).
>
> - I have tried different blocking solution (Rabbit serum 2%, FBS 2%, Horse
> serum and Universal blocker solution from Dako) without any results.
>
> - I have tried different diluents for my antibody (PBS 1X, PBS 1x-BSA 3%,
> Dako diluents)
>
> - I have tried different permeabilization methods (saponin, ptwx) (Not
with
> acetone-methanol fixation).
>
> All I have tried gives always the same signal with a very high intensity
> signal.
>
> More details : I use a biotin-Goat-anti-rabbit for secondary antibody that
> give not a signal if first antibody is omitted. I use PBS to dilute my
> secondary antibody. I use Streptavidin-alexa to detect the signal.
>
> I have also worked on endogen biotin on this cells and it does not appear
to
> be the problem.
>
> Has someone reported this kind of problems on human smooth muscle cells ?
> Has someone suggestions or solutions to this problem ?
>
> Thanks in advance
>
> Bertrand
>
>
> Bertrand Lefort
>
> Research Assistant
> Laboratory of Neuro-immunology of Asthma
> Research Center, Room J3155
> 5400, Blv. Gouin West
> Montreal, QC
> H4J 1C5
>
>
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