For our LMMP protocol, first I placed the tissues in phosphate-buffered saline (PBS; 0.1 M, pH = 7.4) containing niphedipine for 15 min, then the tissues were fixated in 4% PFA in at 4 °C overnight. After 24 h the tissues were washed subsequently for 3 × 10 min with PBS. After that sections of the intestine and stomach seprated under a stereo microscope (mucosa and circular muscle). However, there is suspicion that the tissue is being inverted during mounted of sections with mounting medium after staining. Do you have any suggestions regarding this issue? And also what is the best method by which the myenteric plexus of the intestine and stomach can be isolated from whole mounts for histological staining? In addition is zamboni fixative more effective than PFA as a fixative solution?
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