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Question regarding immunohistochemsitry and metachromatic ATPase staining in mus

immunohistochemistry Metachromatic ATPase Muscle physiology

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#1 LyleBabcock

LyleBabcock

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Posted 23 August 2012 - 07:12 PM

Hope I'm in the right forum....

What I am trying to do is combine an immunohistochemistry protocol (HRP conjugated secondary) with a metachromatic ATPase stain on muscle cross sections so I can look at the muscle fiber-type specific expression of certain growth factors.

Right now both the ATPase stain and the immunohistochemistry stain work perfectly by themselves...

What I have tried so far:
typical ATPase stain followed by a immunohistochemistry protocol. The ATPase stain washed out but there was no interference with the immunohistochem stain

Immunohistochemistry stain followed by ATPase stain. The immunohistochemistry stain worked, but there was no ATPase staining (the blue dye we used stained the coplin jar more than the sample!)

Then I incubated a different slide in all the different buffers I use for the immunohistochemistry protocol before starting the ATPase stain, trying to find if a particular buffer could be singled out. There was major interference in the ATPase stain from all the buffers, especially the plain DPBS(Ca+ Mg+).

And today I again tried an immunohistochemistry stain followed by ATPase, however I used a few washes in DH2O and a few washes in ethanol before I started the ATPase stain. Again, no interference with the immunohistochem stain, but this time the nuclei in the sample were slightly blue but absolutely nothing else (I guess you could call that progress).

I understand pretty well the principles behind both these techniques but I cannot figure out why they would be incompatible. I read a paper recently where they combined histochemistry with a PAS stain, which involved an acid wash, so I'm inclined to think the varying pH between the buffers of the ATPase stain is not an issue.

Any help would be greatly appreciated!





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