Re: Frozen section peroxidase blocking

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From:"Mary Stevens" <>,
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We had a PI who performed IHC on bones fixed with 95% IMS (Methylated spirits.)  He first performed it on decalcified, paraffin embedded samples - with success.  When we supplied the MMA specimens (undecalcified and 95% IMS fixed) he had no success - albeit he only tried for a couple of weeks, and did minimal tweeking.  The same MMA procedures have since been applied to 70%EtOH undecalcified large bones for ISH - with some success.  Results were "muddy" but positive - we will continue to work on it.  So the long winded response to your question is no success to date.  

Good luck- 

>>> Gayle Callis <> - 4/30/2000 9:45 PM >>>
To  keep frozens from being chewed off slides, peroxidase block can be done
several ways.

1.  Dako peroxidase blocker, contains 0.03% H2O2, and some sodium azide,
does not quench blood cells totally, but works great for CD markers.

2.  Glucose Oxidase block is the best, total quench, but more time
consuming, however gentle and thorough

3.  A new thing just learned, but did not find it 100% effective when doing
fixation at 4C or in cold, and want to try it again at RT.  Fix sections in
RT acetone containing 0.5% hydrogen peroxide, for 10 min, fascinating way
to do two things at once! 

As for methanol fixation of bone, with tetracycline label, you could only
try, depending on the antibody, MMA embed and remove the MMA after sections
ala Hand method.  Just a suggestion.

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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