RE: [Histonet] anti-CD11b

From:Gayle Callis

Why not come back with a secondary that is directly labeled, and have NO
biotin in system at all. Mac1 stains so strongly on frozens(that is what he
is doing), he could use goat antiRat-HRP adsorbed to mouse or Donkey
antiRat-HRP adsorbed to mouse, F(ab')2 fragments of IgG preferred to avoid
fc receptor cross reactivity.  Biosource and Jackson Immunoresearch have
these antibodies.   

We have made our own mouse on mouse kits using Scyteks blocking reagents,
same as they use in their kits with similar instructions for use, and then
a secondary that is not biotinylated.  May take more work, but I would do
directly labelled secondary first with Rat antiMouse CD11b to avoid biotin
altogether in problem liver.  
One could even use a FITC labelled rat antiMouse CD11b and come back with
antiFITC, no biotin involved here either. The secondary rabbit antiFITC
from DAKO can be detected by their polymer kit, if I remember correctly.
The joy is a primary useful for both IHC and FACS.  

Chemicon mouse on mouse immunohistochemistry staining kits ARE biotin free
- we tried on and had clean results but we DID use frozen sections. 

At 01:26 PM 5/28/2004 -0600, you wrote:
>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
>-----Original Message-----
>[]On Behalf Of Gayle
>Sent: Friday, May 28, 2004 9:52 AM
>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
>>Histonet mailing list
>Gayle Callis
>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)
>Histonet mailing list
Gayle Callis
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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