Re: Cyclin D1
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From: | Phyllis Davie <pdavie@phenopath.com> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
Hoo-Boy! Wouldn't I love to hear that information too. I have just
placed an order for my 5th clone of Cyclin D1. They all "work"
(sort-of), but none at a level that I would call good. I'll give you a
run-down of what has worked for me, but I would sure like to hear from
others too.
clone P2D11F11 (Novocastra cat# NCL-CYCLINC1-GM) 1:250.
Pretreat by steaming 20 minutes in 6mM citrate, pH 6.0, followed by a 20
minute cooldown. Rinse in water, then digest 10 minutes in 0.1% pronase
(w/v in PBS) at room temperature. Rinse again, then proceed. This
treatment hereafter referred to as S20/+10.
clone DCS-6 (Dako cat# M7155) 1:100 S20/+10
clone 5D4 (Immunotech-Coulter cat# 1740) 1:1000 S20/+10
clone DCS-6 (same clone as DAKO, but from NeoMarkers, cat# MS-210-P0)
1:500, S20/+10
clone AM29 (Zymed cat# AS18-0220) 1:100, Pretreatment in a microwave
pressure cooker in the citrate for 10 minutes at pressure, followed by
cooldown until 60 degrees C.
clone DR1BC2 ( BioCare Medical CM090A or CM090D) ON ORDER (the
company
recommends using their "EDTA Decloaker" antigen retrieval buffer for best
results)
I have tried a wide variety of other pretreatment options with varying
buffers, pH's, times, heat, no-heat, enzymatic digestions, ... anything I
could think of. The most recent thing I heard was that a Tris/EDTA
buffer at pH 8.5 would do the trick, but I don't know concentrations of
the salts. We gave it a try with 100mM Tris and 10mM EDTA for 20 minutes
in the steamer. It gave results that were "working", but no better than
my S20/+10. If anyone knows the magic antibody or pretreatment, I would
dearly love to hear about it.
>Anyone have any recommendations for good results with cyclin D1. Which
>company's antibodies and which method works best for you?
>Thanks in advance, J:>)
>JOYCE WEEMS
>PATHOLOGY LAB MANAGER
>
Phyllis Davie
Clinical Laboratory Supervisor
PhenoPath Laboratories
Seattle, WA
pdavie@phenopath.com
(206)374-9009 phone
(206)374-9009 fax
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