Immunohistochemistry of porcine enteric neurons
Gemma Mazzuoli-Weber, Michael Schemann, Kristin Elfers, Birgit Kuch, Susanne Hoppe
Abstract
This protocol is to investigate the presence and localization of neurochemical markers in the submucosal and myenteric plexus of the porcine colon using immunhistochemical staining techniques.
Steps
Samples of colon were taken from apparently healthy pigs, placed in ice-cold oxygenated Krebs solution for preparation and immediately transferred to the laboratory. Tissues were then dissected in the ice-cold oxygenated Krebs solution for preparation to obtain whole-mount inner submucosal plexus or myenteric plexus preparations.
Tissue specimens were fixed overnight in a solution containing 4% paraformaldehyde and 0.002% picric acid in 0.1 mol/l phosphate buffer and then washed (3 x 10 min) in PBS.
The preparations were then incubated in PBS/NaN33/horse serum for 1h at room temperature followed by 12h and 2h incubation with the primary and secondary antibodies, respectively.
Specimens were washed in PBS, mounted on object slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 containing 65% glycerol.
The preparations were examined with an epifluorescence microscope equipped with appropriate filter blocks. Pictures were acquired with a camera connected to a computer and controlled by Olympus cellSens Standard Software.
Pictures of the stained ganglia were acquired.
