[Histonet] Glucose oxidase

[Veronique Andriessen]

Hi histonetters,

I've got a problem. I'm have to do immunostaining with HRP and AP on
inflamed rat pancreas (fresh frozen sections, fixed with acetone ethanol
3:1). I have to quench the endogenous peroxidase in the white blood cells.
I have tried the glucose oxidase block as described in the histonet
archives, but I am not able to get it working.
I have ordered the correct ingredients from Sigma, but nothing! The only
thing that is not recently purchased is the Sodium azide, but I can't
imagine this going bad. I even tried adding 0.1% saponin, but this also
did't help.
I really need some advice, I'm getting desparate.

Kindest regards,

Veronique Andriessen BAS

Lab. Molecular Liver Cell Biology

Free University Brussels (VUB), Belgium

Tel: 0032-2-477.4259

Fax: 0032-2-477.4412

vandries <@t> vub.ac.be



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 37°C 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.