Problems with VECTASHIELD

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sophiejcoyle's picture
Problems with VECTASHIELD

 I am a medical student currently undertaking a research project in PsyRING labs at University of Glasgow. My background is very clinical and not scientific, so please excuse the probably very un-technical terminology!
Part of my project is to combine WFA Lectin Histochemistry and pCREb immunohistochemistry together on rodent brain sections. 
The WFA technique worked well, and I used Vectashield Mounting Media, and could clearly see the desired result, the same happened for the pCREB technique. 
However, when I combined both techniques, a sort of 'skin/layer' seems to be forming over the slides. I tried the combined techniques again with Vectashield hard set, and the same has happened, although it is not as bad, and there seems to be more 'islands' of sections without this layer over the top. 
My supervisor believes that this must be a result of the mounting medium, although, is unsure why or how. 
Do you happen to know of any interaction between Vectashield mounting mediums and PBS (which I'm using as a wash) that may cause this? 
Or has anyone came across this sort of layer over a slide before and knows why it happened? 
Any help is very appreciated! 


JulietteSV's picture


I have done immunochemistry before and used PBS/Vectashield and never had this kind of  problem. So I don't think it's the PBS...
I don't understand very well what you mean with the "layers", but if the vectashield worked fine when you did the two techniques separately I don't think that's where the problem is coming from. To be sure you can look for an alternative mounting thechnique, or maybe look at publications that have combined histo and immunochemistry...Maybe some steps in your combined protocol are the ones creating the problem.

Sorry I couldn't be of more help!