[Histonet] RE: PhosphoGuard fixative additive

From:"Johnson, Teri"


I have not tried the PhosphoGuard, but I have had some of our researchers here use a fixative recommended by Ventana for phosphorylated proteins. Below is the information in its entirety (apologies for the length).


The phosphorylation state of serine, threonine and tyrosine residues of regulartory proteins control a wide variety of cell functions including cell cycle, growth, apoptosis and signal transduction. Examining the phosphorylation and/or dephosphorylation state of these proteins in fixed tissues may provide insights into cell's response to changes in internal and external environments as well as their impact on disease states. Due to the presence of cellular phosphatases, however, the half-life of many phosphorylated proteins is short and may be underrepresented if tissue specimens are not fixed immediately upon excision in a fixative designed to inactivate cellular phosphatases as well as fix the tissue. Ventana Fixative is a multi-component, formaldehyde based fixative designed to rapidly inactivate phosphatases in cell and tissue preparations. The final cocktail contains sodium fluoride for inhibition of serine and threonine phosphatases, sodium orthovanadate for inhibition of tyrosine phosphatases, sodium molybdate for inhibition of phosphoprotein phosphatases, sodium tartrate for inhibition of acid phosphatases, imidazole for inhibition of alkaline phosphatases, and formalin for rapid cell or tissue fixation.

Instructions for Use

Just prior to use add 200 ul of Fix Prep 1 and 200 ul of Fix Prep 2 (see formulations below) per 10.0 ml 10% Neutral Buffered Formalin (NBF). Mix well. Tissue specimens should be immersed in a minimum of 15 volumes of Fixative for 24 hours and then transferred to 70% ethanol for transport or storage until processing. Cell suspensions should be pelleted and resuspended in a minimum of 3 mls of Fixative for 4-6 hours, and then transferred to 70% ethanol for transport or storage until processing. Tissues should be placed into Fixative immediately upon excision. Any delay in placing the excised specimen into Ventana Fixative may impact the quality of phosphoprotein preservation.

Please carefully read the MSDS instructions and all associated product information provided by the product vendor including safety precautions associated with each raw material and their appropriate use. Latex (or equivalent) gloves, safety glasses, and other appropriate personal protection should be worn when handling the reagents.

Fixative Formulations

Fix Prep I - 0.5M Sodium Fluoride - 2.1g Sodium Fluoride (Sigma Cat. No. S7920) in 100 ml deionized water
Fix Prep 2 - 50% Phosphatase Cocktail 2 - Dilute Phosphatase Cocktail 2 (Sigma Ca. No. P5726) 1:1 in deionized water

Store both at 2-8 degrees C (Do not freeze)

I hope this provides an alternative to try for those who would rather make their own material over buying commercially prepared reagents.

Best wishes,

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

-----Original Message-----
From: ihcrg@googlegroups.com [mailto:ihcrg@googlegroups.com] On Behalf Of Connolly, Brett M
Sent: Wednesday, July 02, 2008 8:46 AM
To: histonet@lists.utsouthwestern.edu; ihcrg@googlegroups.com
Subject: [IHCRG] PhosphoGuard fixative additive

In the June 2008 issue of CAP Today, I ran across a fixative additive called PhosphoGuard from Targeted Molecular Technologies.

They claim it prevents degradation of phosphorylated and activated proteins allowing for better IHC detection (stronger/more abundant signal) by better inactivation of phosphatases compared to plain 10% NBF.  Apparently you just add some to your formalin.

You can check it out further on their website. http://www.tmdlab.com/technologies_PHOSPHO.htm

Just wondering if any of you have tried it?


Brett M. Connolly, Ph.D.
Research Fellow, Imaging Research
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
PH 215-652-2501 fax. 215-993-6803
e-mail. brett_connolly@merck.com
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