Bx3 microscope

Hematoxylin and Eosin (H&E) staining was conducted on frozen TG tissue sections (5μm) to visualize ganglion structure. Briefly, slides were incubated in Harris’ Hematoxylin for 5 min, rinsed in H₂0 for 5 min and differentiated in acid alcohol (1% HCl in 70% ethanol) for 10 sec. Next, sections were rinsed with distilled H₂0 (dH₂0) for 5 min, incubated in ammonia water (0.25% ammonia hydroxide in dH₂0) for 3 min and rinsed with dH₂0 2X’s (5 min). Sections were dipped in alcoholic Eosin for 10 sec, rinsed with dH₂0 2X’s (5min), dehydrated in ethanol and then mounted onto coverslips (Permount, Fisher Scientifc; Hampton, NH).
Protein expression was also determined in frozen TG sections using an antibody directed against glial fibrillary associated protein (GFAP) (Z033429-2, Agilent Technologies, Santa Clara, CA). A standard immunohistochemical avidin-biotin-peroxidase complex technique (Vectastain Elite ABC kit, PK-6100, Vector Laboratories, Burlingame, CA) was used to visualize protein expression using 0.02% of 3-diaminobenzidine tetrahydrochloride. Sections were then counterstained with methyl green. Images were acquired with an Olympus BX3 microscope equipped with a DP73 17.28 megapixel digital color camera using a 20X objective (Olympus Life Science; Center Valley, PA).

Protein expression was determined in frozen TG tissue sections using antibodies directed against TRPV1, CGRP, (GP14100; RA24112, Neuromics, Edina, MN), ET-1, ET-A and SP (ab2786, ab117521; ab14184, Abcam, Cambridge, MA). Following primary antibody incubation, tissue sections were incubated with the appropriate Alexa-Fluor 568 and 488 secondary antibodies (Life Technologies; Grand Island, NY). Sections receiving only secondary antibody were included as negative controls. Immunofluorescence images were acquired with an Olympus BX3 microscope equipped with a DP73 17.28 megapixel digital color camera using a 20X objective (Olympus Life Science; Center Valley, PA).