[Histonet] Glucose oxidase

[Veronique Andriessen]

Thanks for your suggestions Phil, but I've tried all that.

I made the solution fresh and added the GLUOX to the buffer just before
putting it in the 37C water bath. I also tried omitting NaN3 and different
concentrations of GLUOX in the buffer but nothing worked.

Veronique
  -----Original Message-----
  From: Phillip Huff [mailto:histophilhuff <@t> yahoo.com]
  Sent: woensdag 15 september 2004 18:00
  To: Veronique Andriessen
  Subject: Re: [Histonet] Glucose oxidase


  The glucose oxidase procedure requires that the solution is fresh when
used. We make the solution immediately before we need it, warming it in a
water bath (37C) for 5 minutes before use. We currently do not use Sodium
Azide in our reactions so you could try dropping that reagent and make the
solution fresh.

  Good luck,

  Phil

  Veronique Andriessen <vandries <@t> vub.ac.be> wrote:
    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 usefu l 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.

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