[Histonet] uPAR

[Andrea Marion]

Hi Shirin,

It looks like you've tried a number of different heat intensities. Rather
than try different combinations of heating, I think you would do better to
try a different retrieval buffer entirely. Have you tried sodium citrate
at pH 2.5, or 8.0? What about EDTA or enzymatic digestion? Different
epitopes require different conditions. I haven't used the antibody you're
referring to, but a quick search of google turned up a paper that tested a
panel of different antibodies for uPAR against different antigen retrieval
methods. It looks like they get the best results using pH 2.5 0.05 M
sodium citrate, and heating to boiling in a microwave oven, then heating
at 150W for 10 or 20 minutes. Pronase digestion gave better results.

You can read the paper (free access) here:
http://jhc.sagepub.com/content/46/4/469.full

It might also be worth considering whether your positive control tissue
was processed properly, and whether the antibodies you're using have been
validated on FFPE tissues.

Good luck,
Andrea

Andrea Marion
Graduate Student
University of Illinois at Chicago


 Dear histonetters
I'm working on a surface marker, uPAR, on lymph node tissue. For
setting up the IHC-p procedure, I've chosen the ductal carcinoma
sample as positive control. I use the citrate buffer ph=6 as retrieval
solution and TBS ph=7.6 for wash steps. different Ag retrieval systems and
different Ab ( santa cruz biotech. ) dilutions have been tested
s 2*30 minutes of heating in water bath 70 degree of cellicius,
5 minutes with 800 watt power in microwave oven following 15 minutes
heating with power of 300
6 minutes with max power ( 800 watt) following by 15 minutes on power 300
watt
10 minutes with max power following with 15 minutes on power 300 watt
20 min autoclave on 121 degree of cellicius
But there is still no signal on ductal carcinoma sample.
Is there anyone have a comment for me?
I would be so appreciated .


Shirin kouhpayeh
Ph.D student
Immunology dept.
Isfahan university of medical science
Isfahan, Iran