In situ hybridization - (Jan/16/2015 )

Hello Everybody !

I'm new to the forum so here is the story, I'm a PhD student in switzerland for 2 years now and I started doing in situ hybridization.

In order to do so, we use S35 to mark our gene of interest and then we labelled the nuclei with an Hoechst dye. Attached to this Email you will find the procedure we use. I need your help because it seems that for some slides, Hoechst dye failed to work (I might have inverted the racks, as it is in the complete dark and failed to put it in the one containing the hoechst)… When I look at my ISH with the appropriate filter under the microscope, I can see that it worked. But I can't do anything without my Hoechst staining … I would like to know if it is possible to re do my Hoechst staining in order to save my slides and if so, could you please tell me how or at least a hint. :)

I was thinking of redoing the second day entirely but don't know if it is worth it.

Thanks by advance!

Have a nice day,

Bouh68

So you are doing ISH on tissue or mouse embryo sections using 35s labeled probe.

Hoechst helps you in outlining where exactly the tissue (and cells) is by staining all nuclei, but beyond that it has no importance. So, I would suggest that you complete the protocol, including last incubation step of 1 week (I used to do it for longer - even 1 month, as I remember). Then you take your photographs. If your signal has some background, you will see the complete tissue outline. If the signal is very focal, without background, you may not, in that case, jot down the coordinates on your microscope really well.

After that, you restain the slides with Hoechst, give it a much longer incubation (10 minutes) because of all the processing that has gone, and hope that it works, because I have never had to do this (so take my advice with a grain of salt). But at least all your photographs are already taken, so you have not much to lose. I wonder if you can even get by doing something like Methyl green counterstain, if precise signal localization is not a big concern.

Thank you for your response. Yeah my big concerned is to remove this canada balsam… I mean, once it is gone I suppose I can do my dapi/Hoechst staining, I just don't know if the SSC will remove it or not… So I just redo the second day from the 5x SSC at 63C to the last dehydration step?

I'm doing in situ on brain tissues. :)

You must understand I am going out on a limb here. Never done this, but I think it depends on how well the resin (canada-balsam) has already set. Waterbath, direct heating on a hot plate, microwaving, all sorts of things are done to remove resin (especially in museum specimens), but in our context, the best thing to begin with may be xylene. See if you can remove it clean. Xylene should not damage or lift off the tissue. 

As I am not sure, why don't you take a slide with just counter-stained brain section or unstained section, cover them with coverslip in the same manner, let the resin set and try to remove it with various methods? Even your old ISH slides no longer of use would be good for this purpose. 

You don't need to start from day 2. You start by dipping the slides in xylene!

Let us know what happened, even if that means cursing me with choice words :)

I was thinking about something. To remove excess of canada balsam on the coverslip we use a Q tip and acetic acid. It works pretty well. Do you think I could use this or it risks to arm my ISH staining ? 

That could work, although acetic acid may eat up your tissue,  but you just have to try.  

BTW, by doing anything with these slides you are risking your ISH stain (xylene being safest, but not risk-free). That's why I suggest that you first take your pictures very well, including noting down the coordinates if you think necessary, and then only you proceed with the potentially disruptive experiments!