Maya1988

Dear all,

Once again it's me - I am having huge problems with my ISH. I cut the 4%PFA fixed tissues (embedded by myself in 100% clean, fresh, melted paraffin) onto poly lysine slides, dry them at 37 oC overnight and then go through extensive deparaffinization steps with xylenes (all fresh) and ethanols...however, I am getting humongous background!!! All around the sections, exactly in the shape of the original paraffin slice...

I observe this in nearly all my slides however not in ALL of them! How can one slide be completely clean, but so many others are dirty? And no, none of them dried overnight, all of them were treated in the same batch!

Did anyone ever face this problem? What to do? I am really at the end of my wit here and since this is so random and does not occur in all my slides, I don't even know how to best trouble shoot...

T.T

Maya

Dear all,

I am looking to use an antibody for IGF2 for immunohistochemistry and Western blot (mouse). Do you know of any IGF2 antibody which has been shown not to cross react with
- IGF1
- insulin?

Please let me know, or any tips on how to best assay an AB for cross-reactivity are welcome, too! Neutralization assay?

Thanks a lot,
Maya

Hi all,

Please bear with me trying to puzzle out the following problem, it concerns in situ hybridization on paraffin embedded slides:

I am looking for expression of gene A with a probe which I know has worked for gene A previously (in whole embryos).

I know that gene A is highly expressed in the liver, and not very highly expressed in my tissue of interest.

My positive control for my ISH experiments is thus liver with anti sense (AS) probe - works perfeclty and I get a nice strong signal after 2hrs of exposure (I use NBT/BCIP with DIG labelled probe). My negative control, liver with sense (S) probe, is also clean.

However, in the tissue of interest (where expression of gene A is so low that I need to develop overnight), I see an increase of both AS probe signal, and S probe signal across various ages of my mice (not the same strength as AS, but still, the same increase over various ages).

I am really at my wits' end, since I think this can't be probe contamination, as my liver is negative with the S probe? Or can the sense probe be contaminated with AS, and not come up in the liver but in the tissue of interest? I have been doing this again, from scratch, to see if it is still the same, and how repeatable it is, but how can it be a contamination of the S probe with AS if my liver S is so CLEAN?

Please, any ideas/help highly appreciated!

Best,
Maya

Ps. Is there any (tiny) possibility that in my tissue of interest an AS transcript exists of gene A, and I am picking this up with my S probe? Or is this utterly utopic?