Re: [Histonet] About H2O2 and CD Markers

From:Gayle Callis

We prefer to use the least concentration of hydrogen peroxide, and always
in a PBS rather than methanol solvent when blocking endogenous peroxidase
in FROZEN  SECTIONS.  You will find that the higher the concentration of
H2O2, the more likely frozen sections (which is minimally fixed with
acetone or even acetone/absolute alcohol) will be chewed off the slide. The
section was totally worthless with badly damaged morphology. This happened
when we first set up immunostaining. We sawa bubble appear on top of the
section when more concentrated H2O2 blockers are applied.  That led us to
the two blocking methods below. 

We use either the DAKO S2001 blocker that contains buffer, 0.03% hydrogen
peroxide and a bit of sodium azide and is used for 10 minutes, although the
package insert indicates less, we have done 10 to 15 min without problems.
 It is very gentle and effective.  The best blocking is done by a glucose
oxidase method, which preserves morphology in frozen sections AND quenches
peroxidase and pseudoperoxidases completely.  This is by far our favorite
endogenous peroxidase blocker.  

You should try higher concentrations and observe IF your frozen sections
come off the slides or damaged by the peroxide.  When this happens, then
you should consider a gentler method.  Methanol is NEVER used with CD
marker staining, it is known to cause diminshed staining of these markers,
and was discussed by people staining paraffin sections and experienced this
problem. They switched to buffer with hydrogen peroxide.  Methanolic
bridges or cross links occur - this is also found in the literature (Elias

Paraffin sections are much more robust than frozen sections, and paraffin
withstand stronger peroxide concetrations although methanol is still
avoided for murine CD markers.  You can avoid peroxidase blocking entirely
IF you use alkaline phosphatase enzyme methods. 


At 12:08 AM 3/27/2004 +0000, you wrote:
>Hello everybody.
>I have a simple question: do someone know the effect, if any, of the 
>different concentrations, and times, of H2O2 on the CD markers (Paraffin 
>and/or frozen sections)? I have read that some people use 0.01%, others 0.1% 
>and others 3% for different times, usually 10 or 30 minutes. What could 
>happen if I use a longer time?
>Thank You very much.
>Help STOP SPAM with the new MSN 8 and get 2 months FREE*  
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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