Posted 25 September 2011 - 12:26 PM
I have a little bit of problem that I need your help to figure out what is going on.
I have this tissue in frozen block. this tissue is from a transgenic mouse with b-gal reporter.
(tissue was stained with X-gal before cryopreserved, so I can see there are blue cells in the tissue when I made section.)
I was using this tissue section and staining with some antibodies with DAB staining to see the identity of the blue cells.
Then, I just did staining of this tissue with anti-beta-galactosidase (DAB) antibody to make sure DAB staining and X-gal colocalize, but ended up realize some do, but some dont.
I mean that I can see some cells are blue (X-gal+) but DAB- (beta-gal negative), or DAB+ but not X-gal positive.
Does anyone see the similar results before, or know any publication reporting this? or any suggestive explanations for this?
BTW this tissue was isolated from animal,
Rinse with PBS,
Stained with X-gal solution,
Wash with PBS,
Fix with 4% PFA
and wash with PBS and proceeded to cryopreservation.
Any suggestions help, so please let me know
Thanks in advance
Posted 25 September 2011 - 08:18 PM
Posted 25 September 2011 - 08:30 PM
yeah I knew about senescence, and x-gal is normally use to detect senescence, but most likely my cells are not because of that.
Posted 26 September 2011 - 01:24 PM
Posted 26 September 2011 - 03:09 PM
it is a good point, but I think the permeability is not a key, because
I dont do permeablization step perticulary for the staining, but I use PBST for all the staining wash, and
I do use same protocol (with similar tissue) for Sox2 or PCNA, and works fine.
so the tissue should be permeable enough that b-gal antibody to penetrate.
Also, the problem that I cannot figure out is there are cells that are either b-gal antibody positive or X-gal positive only.
maybe this is caused by combination of more than one issue.
Posted 28 September 2011 - 09:57 PM
I am trying to make more samples to see whether I can see the same things or not.
Posted 06 October 2011 - 09:21 AM
what do you think? is this possible???