Re: Immunos on Bone Marrow

From:Mikael Niku

We are using the following buffer for lysing RBCs
(either remaining RBCs in leukocyte fractions or even
for preparing leukocyte cytospins from whole blood):

155 mM NH4Cl
10 mM KHCO3
0.1 mM EDTA

Mix cells in excess volume and incubate for about 5 min
in room temperature (less is enough for removing RBC
contamination in leukocyte fractions, 5 min should be enough
for whole blood).

We got this from Miltenyi Biotec's MACS protocols - but
I guess it's a pretty standard way.

We are using a pretty wide selection of antibodies tested
OK for bovine tissues. Most are from Serotec, VMRD or
DAKO. Feel free to ask more specifically.

About RBC stain: how about just exploiting the endogenous
peroxidase activity with DAB and using another method
for the second staining (for example, alkaline phosphatase;
there are several substrates with different colors to choose from).

   Mikael Niku             URL:
   University of Helsinki  Dept. Basic Veterinary Sciences
       - Mitäkö mieltä olen länsimaisesta sivistyksestä?
         Minusta se olisi erinomainen ajatus!
                                              - Gandhi

----- Original Message -----
Sent: Thursday, November 01, 2001 2:02 AM
Subject: Immunos on Bone Marrow

> Does anyone have experience doing immunos on bone marrow, cytospins from
> bone marrow or smears?
> Due to the large number of red blood cells, it is difficult to see
> stained cells
> using DAB. Is there  another way to lyse RBC besides methanol/peroxide?
> I am trying to stain for macrophages in one satining procedure.
> I also need to stain erythrocytes. I need to do a double
> staining method on the erythrocytes with co-localization within the same
> cells so I need to use 2 different colors.  Any suggestions?
> Also, what is the best sources of anibodies for animal tissues?
> Thanks in advance

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