Re: MIB1

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From:Mary Vaughan <mvaughan@sc3102.med.buffalo.edu> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Dear Heike,
        MIB-1 [Ki-67] took a little extra time.... but I managed to get the
assay working. I've not done bone marrow, but even "regular"  soft tissues
[form fix - paraffin] required an extra effort. The literature pointed in
this direction also, although I don't have the references at my fingertips.
        Temperature and duration are two very important factors here. This
is the only Ab [in my lab]  that requires double time in the microwave
{citrate pH 6.0}. My usual pre treatment time is 10 min and with this
Antibody, 2 X 10 min worked nicely. Also, I used an overnight incubation
with the primary @ 4 g/ml. I tried 6 different pre treatments, and nothing
else worked. {Don't forget your negative control}. Your secondary system
sounds OK. Let me know if you have any specific questions.

At 10:14 PM 3/4/99 -0800, you wrote:
>Histonetters!
>anybody out there doing immunohistochemistry on bone marrow sections with
>MIB 1? We get no staining so far. However, I have to admit that in
>Germany the routine fixation for bone marrow biopsies contains a
>combination of glutaraldehyde, formaldehyde and other ingredients. So, I
>currently assume it is the glutaraldehyde which destroys the MIB1
>epitope.
>But may be, there are some suggestions aout there? some special
>pre-treatment? We use a pressure cooker (5 min) in citrate buffer at the
>moment and a straeptavidin-biotin-hrp- detection system.
>
>thanks for your help
>
>Heike Grabsch, Germany
>
>
				Best Regards,
				Mary Vaughan HT(ASCP)
				Roswell Park Cancer Institute
				Pharmacology + Therapeutics
				Elm + Carlton Sts.  CDC-121
				Buffalo, NY 14263




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