7 replies to this topic

[Rnotk]

Hey guys,

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

[bob1]

It may not apply in tissue, but senescent cells can utilise X-gal and turn blue when stained like this. I don't think this pathway uses B-galactosidase.

[Rnotk]

Thanks bob1 for the reply

yeah I knew about senescence, and x-gal is normally use to detect senescence, but most likely my cells are not because of that.

Thanks though

[bob1]

Did you permeablise the tissue after fixing? If not, it might be that you have some cells that won't have taken up the antibody because they are still impermeable.

[Rnotk]

bob1 thanks again

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.

[bob1]

Antigen recovery?

I'm running out of ideas here.

[Rnotk]

yea, me neither,

I am trying to make more samples to see whether I can see the same things or not.

Will see

[Rnotk]

it has been suggested that X-gal staining process before fixing tissue might affect the epitope integrity. (since I do X-gal staining of the tissue then fix the tissue with PFA, so some epitope (b-gal) might be still X-gal positive but not stained with antibody)

what do you think? is this possible???

Thanks