[Histonet] RE: alternative to Glucose oxidase (GLUOX) peroxidase block

[gayle callis]

You wrote: 
 
I have a protocol that requires GLUOX but I do not have the chemicals to
make it at this present time. Is there an alternative or can I just use H2O2
at a weaker concentration. The antibody is a home-brew RNF5 on frozen muscle
sections. Any thoughts would be appreciated.
 
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If you are fixing the muscle frozen sections with a solvent e.g. acetone,
then you can try using a very weak solution of hydrogen peroxide in buffer.
Try 0.1% Hydrogen peroxide, and if that eats the sections off the slides
(you will see bubbles coming off the top of the frozen section, particularly
if the tissue has lots of red blood cells, endogenous peroxidase.  You can
always cut the H2O2 concentration down, or even try slightly higher
concentration (0.2 to 0.3%) This has been discussed in the past on Histonet,
and can be found in Histonet Archives. 
 
 I will send you a weak endogenous peroxidase blocker under separate private
email  This is well known and found in the literature.  The solution has low
concentration H2O2 and sodium azide in buffer.  It is a nice inhouse
protocol. 
 
You might have to apply it twice, 10 minutes then drain and do the block
again for another 10 minutes but it does NOT eat you sections off the slide.

 
The alternative is use Alkaline phosphatase instead of HRP then a peroxidase
block is NOT needed. There are some sensitive AP chromogens that are as good
DAB.   
 
Gayle Callis
HTL/HT/MT(ASCP)
Bozeman MT 59715