Dear all, Hope as a community your are doing fine. I have a question regarding antibodies against gH2AX, XRCC1 and p53bp1 that have worked for tissue detection for either IHC or IF, using cryopreserved material or paraffin. And if you truly have one that works, can you please share the protocol? Thanks in advance for your help!
I follow this protocol for my gH2AX staining on parafin embedded tissues and it works.
Protocol:
· Freshly cut 4μM paraffin-embedded sections were deparaffinized/rehydrated by 3 times with 100% xylenes for 5 min each, 2 times with 100% ethanol for 5 min each, 95% ethanol for 3 min and 70% ethanol for 3 min. Samples were rinsed with PBS for 5 mins.
· Antigen unmasking step was performed by boiling the samples in 1mM EDTA for 30 mins and cool down the samples for 30 mins in the boiled 1mM EDTA buffer at RT.
· Keep the samples in 1% Triton X prepared in PBS for 30 mins (gentle shaking).
· Samples were blocked in 10% serum with 0.5% Tween-20 in TBS for 1hr (gentle shaking).
· Primary antibody incubation was done with monoclonal H2AX antibody (1:100) (cell signaling 9718S) for overnight at 4°C (gentle shaking) by adding 100uL of H2AX antibody solution (1% serum + 0.5% Tween20 in TBS) on the tissue and cover the slides with parafilm. Keep the samples in a humidified chamber.
· After primary incubation, samples were washed 3 times with TBS containing 0.5% Tween-20 and then incubated with Donkey anti rabbit secondary antibody Alexa Fluor 488 (1:1000) for 2 hr at RT (gentle shaking). Add 100uL of secondary antibody solution (1% donkey serum + 0.5% Tween20 in TBS) on the tissue and cover the slides with parafilm. Keep the samples in a dark humidified chamber.
· Samples were washed 3 times with TBS containing 0.5% Tween-20 (TBST) in the dark.
· Mount the slides with Vectashield mounting medium containing DAPI and cover with glass coverslip. Add a drop of mounting medium on the tissue and put a coverslip on it.
· Incubate the samples at RT in the dark for 30 mins.
· Store the samples at 4°C (short term storage) or -20 °C (long term storage).
Thanks! When doing the antigen retrieving step, do you boil by pressure cook or with microwave? Thanks again for your helpful protocol! :-)
Elizabeth Pavez Lorie We usually do the antigen retrieval step in microwave using power level 5.
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