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A little info from some of our people in Germany:
Regarding MPO:
A. Fix & Perm is not the only way, but it is a good and easy way (it is
often recommended in consensus protocols)! You can buy it from caltag
in U.S. Most of our customers use this reagent! But it wont work for TdT
staining!!
B. MPO staining should also work by "handmade" methods:
a) e.g., Paraformaldehyde / Ethanol (PFE) treatment; 2x10to 6 cells were
fixed in 1 ml 1%PFA for 15 min.at RT. After that 1 ml PBS/BSA1% were added
drop by drop. Cells then were pelleted and resuspended in 2 ml 45% ethanol
in
PBS and incubated for 30 min. at RT. Then cells were washed twice with
PBS/BSA1% and stained.
- this method should also work, but I do not have any experiences about
background staining.
C. An easy way for reducing background staining is to include a
pre-incubation step with a mouse isotype-control antibody, not
fluorochrome conjugated for 15 min. before the "real" staining starts. Some
of our
customers do this in case of Ki-67 staining.
Jessica
DAKO GmbH
Ellen Ko
DAKO Corporation
Technical Service Specialist
http://www.dakousa.com
(800)424-0021
----------
> From: Elaine Kunze <mek4@psu.edu>
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: Myeloperoxidase reference
> Date: Tuesday, March 18, 1997 6:48 AM
>
>
> Myeloperoxidase reference:
> "Flow cytometric detection of intracellular antigens for
immunophenotyping
> of normal and malignant leukocytes" K Groeneveid, et al. Leukemia
(1996)
> 10, 1383-1389.
>
>
> ************************************
> Elaine Kunze
> Flow Cytometry......Image Analysis
> The Biotechnology Institute for Research and Education
> 8B Althouse Lab
> Penn State University
> University Park, PA 16802
> (814) 863-2762
I have worked with Fix and Perm in the past with MPO and Tdt staining
utilizing single and dual staining and have had good results. These
tips may help:
a)Tdt staining : increase the Ab incubation time up to 1 hour.
b)MPO-you need to block for non-specific binding(20ul AB serum will
work). This is especially important if the sample contains >20%grans
since these will leak out lactoferrin and MPO and may cause an
artificially high % of MPO.
c) Most important- you MUST titer out the appropriate Ab amount that will
provide you with good staining and low non-specific binding.
Sherri Scheirer-Fochler
Bone Marrow Transplant
UPMC
schesh@novell1.dept-med.pitt.edu
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web