RE: Rat Macrophage - Staining Problems

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From:"Hewlett Bryan (CMH)" <HEWLETT@EXCHANGE1.CMH.ON.CA> (by way of histonet)
To:histonet <>
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10-20 micrograms/ml sounds a little high to me, we seldom go above 5 and
with HIER our range is usually 0.1- 2.0 micrograms/ml.
Am not familiar with the antibody you use.

We use mouse anti-rat monocyte/macrophage clone ED-1 from Serotec
cat#MCA341. For formaldehyde fixed paraffin sections of rat lung, we use
HIER in 0.01M citrate buffer pH6.0 for 10 min.@ 96C. ED-1 is diluted
1:250 in 1% v/v normal swine serum in 0.05M TBS pH7.6, incubation is 1hr
@RT, detection is with biotinylated rabbit anti-mouse( absorbed against
rat) (Dako code E0464, diluted 1:300 in 1%v/v NSS/TBS, 1hr@RT), followed
by Streptavidin/Pox(Dako code P0397, diluted 1:600 in NSS/TBS, 1hr@RT).
Chromogen is AEC, if you use DAB you may find that the primary can be
further diluted to 1:500 and if you use intensified DAB then 1:1000!


>From: 	Dave Tacha[]
>Sent: 	October 1, 1998 8:13 PM
>Subject: 	Rat Macrophage - Staining Problems
>We been using a "mouse anti-rat macrophage" from Pharmagen.  We
>incubated the rat tissue (lung) for 1 hour at 10 and 20 micrograms per
>ml.  I also pretreated the tissue with and without antigen retrieval.  I
>used Pharmagen's goat anti-mouse with minimal cross-reactivity to rat.
>However, we are not getting any staining.
>Could someone help me with a procedure, or technical hint; or another
>perhaps another rat macrophage antibody that works in formalin-fixed
>paraffin embedded tissues?
>My main concern is that the secondary antibody may not recognize the
>primary antibody.
>Has anyone ever heard of MOMA?

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