RE: [Histonet] Immunocytofluorescence on smooth muscle cells

From:"Phil Bergin"


We get a lot of unspecific binding in gastric smooth muscle cells and
throughout the whole stomach (both human and murine) when we use rabbit
antibodies, which is really difficult to block.  Basically we try and
stay away from rabbit antibodies as they seem to 'stick to everything'
as far as our lab goes............

Best of luck to you though,


Philip Bergin
Göteborg University
Department of Medical Microbiology and Immunology
Box 435, SE-405 30 Göteborg, Sweden
Phone: +46-31-3424471
Fax: +46-31-826976

-----Original Message-----
[] On Behalf Of Katri
Sent: den 30 september 2003 01:46
To: Bertrand Lefort;
Subject: Re: [Histonet] Immunocytofluorescence on smooth muscle cells

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
cells and I don't know if there is a way to prevent it.

Hope this will help a bit.....


Katri Tuomala
Anatomic Pathology
St Joseph's Health Care
Hamilton, Ontario, Canada
----- Original Message ----- 
From: "Bertrand Lefort" 
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
> in the lab.
> We are working with human bronchial smooth muscle cells.
> I have currently a very big problem with all rabbit antibodies. All
> antibodies (including IgG as isotype) give a non-specific signal,
> 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
> and kit like permeafix).
> - I have tried different blocking solution (Rabbit serum 2%, FBS 2%,
> serum and Universal blocker solution from Dako) without any results.
> - I have tried different diluents for my antibody (PBS 1X, PBS 1x-BSA
> Dako diluents)
> - I have tried different permeabilization methods (saponin, ptwx) (Not
> acetone-methanol fixation).
> All I have tried gives always the same signal with a very high
> signal.
> More details : I use a biotin-Goat-anti-rabbit for secondary antibody
> 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
> 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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