How to Block Endogenous Peroxidase for DAB staining ?

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Kisspep's picture
How to Block Endogenous Peroxidase for DAB staining ?

I recently attempted my first DAB staining in the mouse brain. I used 40um thick sections from frozen brains and we used a "free floating" method. Some protocols advised to use 0.3% H2O2/PBS to quench endogenous peroxidase and others suggested 0.3% H2O2/100% methanol. So I used both. What I noticed is that those that were treated with the methanol solution appeared very "bubbly" whereas those in the PBS treatment were clear and neurons and structures were clearly visible.
Was my experience with the methanol due to some technical error on my part or is this a normal occurance for this tissue and I should use PBS instead ?

Arvind Singh Pundir
Arvind Singh Pundir's picture
 methonal may be harmful to

 methonal may be harmful to some antigens for antigens sensitive to methanol and frozen sections use PBS containing  0.3-3% H2O2 it works best for quenching the endogenous peroxidase.

g a
g a's picture
Hi Kisspep

Hi Kisspep

Methanol induces precipitation of proteins that may may have lead to the bubble like appearance. Im not sure but it May not be recommended for use with brain sections.