Re: Alpha Smooth Muscle Actin Incubation

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To:Nima Farsinejad <>,
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     Hi Nima!  My understanding is that you can incubate antibodies
overnight at 4 C in many cases to increase the antibody-antigen interaction
hence increasing the final signal.  I routinely had to do this with the
more fussy antibodies like CD4 and CD8 in frozen tissue.  In the case of
alpha smooth muscle cell actin it may depend on the tissue of interest and
the fixative.  In the past I would incubate this antibody overnight at 4 C
when the application was on mouse tissue.  The antibody is a monoclonal
thus mouse on mouse can be rather troublesome with background and
non-specific staining issues.   In the case of alpha smooth muscle cell
actin I had superior results when incubating the antibody overnight because
the cooler temperature seemed to decrease the amount of non-specific or
background stain, especially in the adventitia of vasculature.
Additionally I found the 1A4 clone to be particularly sensitive to fixation
and fixation time. I had used both 10% NBF and 4% Paraformaldehyde as
fixatives and had to adjust my protocols accordingly. In the end I had 4
different protocols depending on the tissue of interest, the fixative and
the fixation time. On the other hand, I also applied this antibody to
porcine tissue and could easily acheive good results with just a 30 minute
incubation at RT.
     Overall, each researcher should establish a working protocol for their
own laboratory.  The literature is a good starting point but in the end you
will probably need to find, through trial and error, a protocol that works
best for you. I hope this information is helpful!  Best of luck!

Jennifer Hoover
Eli Lilly and Company

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