Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

“…The solution had been derived from the stripping buffers that are routinely applied to nitrocellulose or polyvinylidene difluoride membranes for re-probing western blots with multiple primary antibodies. The present work and our recent studies [ 4 ],[ 13 , 20 ] showed that this technique also works effectively in wholemount preparations of human colon. In our hands, the method has been tested with up to 9 layers of labelling of wholemount preparations of human gut tissue.…”

“…This resistance to elution was exploited in a recent study in which all enteric nerve cell bodies were labelled with the pan-neuronal marker HuCD, visualised with biotinylated secondary and AMCA-coupled streptavidin. The cell bodies could then be individually identified in 5 subsequent layers of labelling in which 12 selective cell body markers were applied, providing a detailed chemical coding profile of each neuron [ 4 ].…”

“…In wholemount preparations of enteric plexuses this can be achieved by the application of HuC/D antisera, which label nearly all enteric nerve cell bodies [ 13 , 24 ]. Individual cell bodies can then be reliably re-identified by size, shape and location in ganglia, relative to other cells [ 4 ]. The use of biotinylated secondary antiserum to reveal HuC/D can facilitate this process by making HuC/D labelling resistant to elution so that it persists between layers of antisera without requiring re-staining.…”

“…Notwithstanding the power of single cell transcriptome analysis [ 2 ], the “chemical coding” of a neuron (i.e. the combination of neuronal markers that it expresses), has been extensively used as a basis for characterising neurons in both central [ 3 ] and peripheral nervous circuits [ 4 ]. Typically, this is achieved using multiple immunofluorescence labelling to assess co-existence of markers.…”

“…Using this modified protocol, 2 layers of antisera were applied to the same preparation, allowing co-localisation of 5 immunohistochemical markers in each myenteric nerve cell body [ 13 ]. In a more recent study, 12 markers were quantified in over 2500 myenteric neurons in wholemount preparations as a basis for dividing them into classes [ 4 ]. Here we report a series of 7 experiments designed to whether 2-ME/SDS works in wholemount preparations of human colonic myenteric plexus, or whether its utility is restricted to paraffin sections examined in previous published studies.…”