[Histonet] anti-CD11b

[Patsy Ruegg]

Gayle,
CD11b works fine on frozen sections but my investigator wanted to use it on
mouse liver bile ducts ffpe, I tried vigoursly using the m on m staining
system but one of the problems was that it was not frozen to begin with, and
another major problem I could not over come was that there is so much
endogenous biotin in mouse liver I could not ever block enough to get around
non-specific binding (even in frozens) of the biotin.  Does anyone sell a m
on m IHC staining system that does not use biotinylation?  Something like
the labelled polymers would be great.
I realize that the cd68 was anti-human but it did work on some of the mouse
samples.
Patsy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Gayle
Callis
Sent: Friday, May 28, 2004 9:52 AM
To: pengbw <@t> sjtu.edu.cn; Histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] anti-CD11b


Patsy wrote:

I have although had some limited success with DAKO CD68 PGM1
clone which only stains macrophages and monocytes and not all the other
myleoid cells, but it is not easy and inconsistant in my hands.

Caveat:  This is a mouse antiHuman CD68 for macrophages, and you will run
into the problem of staining with mouse antibody on mouse tissue, a whole
other problem introduced requiring mouse on mouse kits and adds to expense
of procedure.   You can avoid this problem by using CD11b aka Mac1 (Clone
M1/70) Rat antiMouse Monoclonal from BD Pharmingen. Do a dilution panel
starting at 10 ug/ml, normally you can have good staining at 1:500 - 1:1000
or more depending on the chromogen used, primary has original conc of
0.5mg/ml. and incubated for 30 min at room temperature.  Be sure to use the
isotype matched control or rat IgG as a negative control  We have superb
success with acetone(75ml/absolute ethanol (25 ml) fixation.  Make sure you
air dry the frozen sections OVERNIGHT AT ROOM TEMPERATURE, then fix in this
fixative for 5 min at Room temperature, do not dry after fixation but go
immediately to 3 changes of PBS (Dulbeccos) and proceed to other blocking
steps (DAKO peroxidase block for frozen sections), biotin blocking
(Vector), followed by IHC.

For a secondary antibody, Goat antiRat bioitinylated, adsorbed to mouse and
is an F(ab')2 frag, from Biosource or a Donkey antiRat biotinylated,
F(ab')2 frag of IgG adsorbed to Mouse biotinylated from Jackson
Immunoresearch is recommended. This secondary should be diluted in the
normal serum block containing mouse serum (mouse serum concentration varies
from 1 - 5%, we take the middle concentration of 2.5%).

We also prefer to use this antibody biotinylated - this eliminates the
secondary antibody, and you go directly to Strepavidin-HRP for 20 min.  We
do not use kits, and our Strepavidin-HRP comes from Biosource (0.5 to
0.6mg/ml diluted 1:500).

For a block, we prefer normal serum 10% goat or Donkey (matched to host of
secondary) with 2.5% mouse serum (heat inactivated) -

Our morphology is excellent, better than acetone fixed frozen sections, and
our results are very clean, without any background staining.  Make sure you
positive control is from an animal stimulated to produce macrophages in
order to control the chromogen staining with a microscope.

 At 09:18 AM 5/28/2004 +0800, you wrote:
>Thanks you all,
>In fact, I\'m going to do it on frozen sections.
>The most prior component I want to stain is monocytes.
>So maybe I should check DAKO\'s mAb mentioned by Patsy.
>
>Baowei Peng
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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