I have the following problem:
I'm doing a usual DAB staining (1°Ab, 2° Ab conjugated, HRP and Tyramide and then DAB for chromogen detection) that works well.
My issue is that many of the TMA samples fell off the slide during staining procedure as well as I have many "DAB dirt" accumulating on top of my slides (I use slide racks with plastic slide holders) and thus preventing optimal penetration of the DAB and efficient washing afterwards.
I do deparaffinzation 10min at 60°C followed by 3x Xylen, 3x 100% EtOH, 1x 90% EtOH, 1x 70%EtOH, 2x dH2O (each 1min).
Dos anyone have any advices for me?
Thanks in advance!
Christophe
Thanks for your response.
Yes the tissue sections fall apart, or fold themselves.
What do you mean by coated slides? The slides come from paraffin-embedded blocks containing an agarose matrix (see: Pu et al., JHC, Volume 55(1): 21–24, 2007)
It is actually a good idea to filter the DAB (I use indeed the pellet), but it won't solve the problem of tissue falling apart.
I tried once tu heat the slides at 60°C for 5min after the 90% EtOH bath, as suggested by our histology facility, but it didn't help.
Hi, For preventing of the tissue section detachment from glass slide, I agree with those two suggestions. Moreover, after DAB incubating, to avoid DAB precipitation covering you section, you have to do a washing step well by PBS or DW for 3-5 times, 5-7 min each. Good luck!
Thank you all for the suggestion. I will try them!
BR,
Christophe
I would recommend not baking the slides, because this can damage the antigens. However, if you use poly-L-lysine or Superfrost coated slides it's not necessary, as long as the slides are dried at 37-40 degrees. I use this procedure for TMA and no sections come off.
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