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DNAse treatment

DNAse post-treatment for nuclear antigens

Rationale:

The use of DNAse to improve nuclear antigen staining has been published long before the AR era 1, 2.
DNAse treatment is currently suggested as an unmasking technique for incorporated BrdU nucleotides 3.
However, BrdU detection in dewaxed, fixed tissues with DNAse treatment leads to poorly reproducible results. BrdU instead is readily demonstrated after conventional AR treatment (EDTA 0.01M, pH 7.5).
This because AR exposes the artificial epitope by separating it from the DNA strand, and does it more efficiently than DNAse alone. This may also enhance DNAse penetration in fixed and embedded tissue.
We found the combined use of AR and DNAse treatment was additive. The strongest anti-BrdU staining was obtained after the combined use (Fig).
Other nuclear antigens (Cyclin D1, Rag-1, Rag-2, Blimp-1) were enhanced by post-treatment with DNAse.
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Procedure:

Use Deoxyribonuclease I (DNAse type II; Sigma, St.Louis, MO USA: D-4527) at 5 U / mL in TBS, pH 7.5
It is convenient to make 10x aliquots (50 U/ml) to be stored at -20C

Pre-warm the incubation chamber by incubating at 37 C or by carefully microwaving (approx 20 secs).

The sections, which have been previously antigen retrieved and cooled, are transferred to warm TBST.

Blot the slides without drying and apply 100Ál or more of the DNAse final solution.
Incubate at 37 C for 30 min.

Wash twice with TBST and proceed with the immunostain.