[Histonet] RE: Ki-67/PHH3 double staining

[Anatoli Gleiberman]

Ronnie,
I don't have any problems with 4-color fluorescence staining using mouse monoclonal Ki67 conjugated with AlexaFluor488 from BD Pharmingen and rabbit monoclonal PHH3 conjugated with AlexaFluor 555 from Cell Signaling (I also added for complete set rabbit monoclonal gammaH2A.X conjugated with AF647 from Cell Signaling and DAPI). All four colors work good. There was no any masking for PHH3. It looks like chromosome staining for Ki67 was a little less crisp than in case when I used combination with antibody against non-phosphorilated H2A.X histone instead of Ki67. So, probably there is a little masking of Ki67 rather than PHH3.

Anatoli Gleiberman, PhD
Director of Histopathology
Cleveland Biolabs, Inc
73 High Street
Buffalo, NY 14203
phone:716-849-6810 ext.354
fax:716-849-6817
e-mail: AGleiberman <@t> cbiolabs.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Houston, Ronald
Sent: Tuesday, June 14, 2011 11:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Ki-67/PHH3 double staining

Has anyone had success double staining these two antibodies for IHC? Ki-67 tends to mask most of the PHH3 staining irrespective of which antibody we use first. Individually, both antibodies have clean and crisp localization. I have seen pictures in articles and on-line of results but am far from attaining anything like these.
We are using the Refine DAB and AP Red kits from Leica on the Bonds. Ki-67 is from Leica and PHH3 from Cell Marque

Any help or suggestions welcome
Thanks
Ronnie

Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager

ChildLab, a Division of Nationwide Children's Hospital

www.childlab.com


700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>
www.NationwideChildrens.org<http://www.NationwideChildrens.org>

"One person with passion is better than forty people merely interested."
~ E.M. Forster



----------------------------------------- Confidentiality Notice:
The following mail message, including any attachments, is for the
sole use of the intended recipient(s) and may contain confidential
and privileged information. The recipient is responsible to
maintain the confidentiality of this information and to use the
information only for authorized purposes. If you are not the
intended recipient (or authorized to receive information for the
intended recipient), you are hereby notified that any review, use,
disclosure, distribution, copying, printing, or action taken in
reliance on the contents of this e-mail is strictly prohibited. If
you have received this communication in error, please notify us
immediately by reply e-mail and destroy all copies of the original
message. Thank you.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

 


This communication may contain privileged information.  It is intended solely for the use of the addressee.  If you are not the intended recipient, you are strictly prohibited from disclosing, copying, distributing or using any of this information.  If you received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy.  This communication may contain nonpublic information about individuals and businesses subject to the restrictions of the Gramm-Leach-Bliley Act.  You may not directly or indirectly reuse or redisclose such information for any purpose other than to provide the services for which you are receiving the information.