Hello everyone!
I performed a TUNEL assay simultaneously with an immunofluorescence (SOX9 antibody) on the same sections of human testis. The fluorescence of TUNEL turned out well, but that of SOX9 did not (no fluorescence), although in the past this antibody has not given problems in immunofluorescence-only experiments . I use the "In situ cell death detection kit, fluorescein" [Roche], following the protocol of the datasheet.
Usually in immunofluorescence-only experiments I use a TRIS-EDTA-CITRATE buffer (ph 7,8) as antigen retrieve, but for this TUNEL+immunofluorescence experiment I used DAKO (ph 6). Perhaps this is the problem.
So.. The question is: has anyone successfully used TRIS-EDTA-CITRATE buffer as antigen retrieve in TUNEL+immunofluorescence experiments? Unfortunately, I cannot afford to "waste" material on testing. Thank you all!
Dear Irene!
Please You look at following article and protocols:
Article Sox9 overexpression in uterine epithelia induces endometrial...
Immunostaining
TUNEL analysis
Dear Alexandr Chernov ,
thank you for your answer!
But in this paper the authors perform immunofluorescence or TUNEL assay in separate experiments.
Instead, I need to perform the immunostaining for SOX9 and the TUNEL assay on the same section at the same time.
For this reason I need to know if TRIS-EDTA-CITRATE (which I use for immunofluorescence) is good as antigen retrieve also for TUNEL.
Thanks anyway!
Unless samples are grossly over fixed the TUNEL generally needs minimal to none antigen retrieval so you need to focus on optimizing your conditions for SOX9 staining. I would suggest using different less valuable human tissue sections to optimize the staining protocol for instance skin biopsy can have SOX9 signals within hair follicles and in the growth area.
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