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ISH - signal in sense - specificity or contamination problem?

in situ hybridization

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#1 Maya1988

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Posted 10 July 2012 - 03:08 AM

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?

Edited by Maya1988, 10 July 2012 - 03:13 AM.


#2 alexa594

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Posted 10 July 2012 - 02:12 PM

Are you certain that the S probe is signal and not background? Sometimes when I do section in situs I will get some background, especially if my tissue became dry at any point during the hybridization step. Did you let the S probe go longer than 2 hours in the liver? If that is the problem one way to combat this is to add extra hyb (how much hyb are you using? I ended up increasing my hyb from 65 ul to 200 ul per slide while keeping probe per slide the same and that really helped to reduce my background).

#3 Maya1988

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Posted 11 July 2012 - 05:55 AM

Hi alexa594,

I have not left the liver S overnight so I can't be 100% sure that I wouldn't have ended up with some background there, too. However, none of the slides which I left incubated overnight dried out (at least not obviously so) - when I stopped incubation the next morning, all of the sections were still completely wet (I circle with pap-pen and actually add 400ul of hyb buffer with probe to each section). So I am quite sure this is not simply unspecific background due to drying out of sections, especially since both AS and S seem to selectively stain specific cell types only within the tissue of interest.





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