Total quenching of peroxidase and pseudoperoxidases, in RBC's

From:Gayle Callis

If RBC's and other endogenous peroxidases/pseudoperoxidases are a problem,
the glucose oxidase method is far superior to take them out. It works on
acetone and/or paraffin sections, although I have not used it on the
latter, that was passed on from Carrie Kyle Byrne.  It is wonderful for not
chewing up frozen sections and really cleans up a lot of diffuse background
plus specifics. It was originally used for IHC on Eosinophils in frozen
sections.  

GLUCOSE OXIDASE BLOCK FOR ENDOGENOUS PEROXIDASE/PSEUDOPEORXIDASES IN
FROZEN/PARAFFIN SECTIONS

Reference:  Andrew SM, Jasani B.   An improved method for the inhibition of
endogenous peroxidase non-deleterious to lymphocyte surface markers.
Application to immunoperoxidase studies on eosinophil-rich tissue
preparations.  Histochemical Journal 19:426-30, 1987

All forms of endogenous peroxidase may not be inhibited by the usual
methanol/hydrogen peroxide or buffer/hydrogen peroxide blocking mixtures.
This method produces nascent hydrogen peroxide that is preferable to the
normal methods using preformed hydrogen peroxide (which you add or buy in
your endogenous peroxide blocker).  This new method to block 'peroxidatic
activity' is consistently complete. This method is an enzymatic reaction
that produces a slow, steady, very low concentration of hydrogen peroxide.
It is particularly useful for monoclonal antibody staining on frozen
sections that are minimally fixed with acetone.  

Procedure:

Glucose (Sigma G 5250) 			0.180 g
Glucose oxidase (Sigma G 6641)  		0.005 g
Sodium azide 					0.0065g
DPBS 						50 ml

This procedure has doubled concentration of all reagents used in original
reference.

Blocking Protocol

1.	Incubate sections 1 hour at 37C waterbath.  You do no have to prewarm
buffer mixed with reagents 		before immersing slides.  
2.	Rinse in DPBS 3 changes/5 minutes each
3.	Proceed with immunostaining

Preweigh and freeze down aliquots of glucose oxidase calculated for 100 mls
working buffer, bring out, and dissolve in DPBS/sodium azide buffer,
immerse slides, incubate, rinse, etc. 

We make up large quantity of DPBS with azide to save time.  Sodium azide is
toxic to you, safer this way.  Keeps for months!   We labet this Glucose
Oxidase Buffer.  






Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu




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