All IHC steps were performed using a 24-well plate on a shaker at room temperature (~24 °C) in dark environment. The IHC steps include washes, blocking, primary antibody, secondary antibody, and were carried out in different wells. Samples were washed 6x5 minutes in 0.1 M PBS (pH = 7.4) and immersed in blocking solution (0.1 M PBS containing 2% bovine serum albumin, 10% normal donkey serum, 2% Triton X-100, and 0.08% sodium azide) for 5 days. After that, the stomach was incubated in primary antibodies (Anti-CGRP Mouse Abcam, Cat#ab81887 at a concentration 1:170 - RRID:AB_1658411) for 5 days in a 0.1 M PBS solution contains 2% Bovine serum Albumin, 4% normal Donkey serum, 0.5% Triton X-100, and 0.08% Sodium azide. The tissue was extensively washed 6x5 minutes in 0.1 M PBS containing 0.5% Triton X-100 (PBS-T) to wash any unbound primary antibody, followed by incubation in fluorescent secondary antibodies (Alexa Fluor 488 Invitrogen, Cat#A21202 at a concentration 1:90 - RRID:AB_141607) for 3 days in 0.5% PBS-T. Negative controls were prepared from stomach without the addition of primary antibodies to exclude secondary antibodies nonspecific binding.
The tissue was washed 6x5 minutes in 0.1 M PBS and mounted serosal side facing up on slide. The tissue was flattened by applying pressure from lead blocks weights (6.75 kg) for 4 hours and dried under the fume hood for 1 hour. The tissue was dehydrated using four increasing concentrations of ethanol (75%, 95%, 100%, and 100%), 4 minutes each, followed by 20 minutes of xylene to render the tissue transparent. DEPEX mounting media was applied over the tissue and covered with a cover slip. The tissue was air-dried in the fume hood overnight.