To visualize the vagal fibers labeled with dextran biotin, free-floating whole mounts were treated with a hydrogen peroxide:methanol block (1:4) to quench endogenous peroxidase activity, soaked for 3–5 days in a PBST (PBS containing 0.5% Triton X-100 and 0.08% Na Azide) to facilitate penetration, and then incubated for 1 h in avidin-biotin-horseradish peroxidase complex (PK-6100; Vectastain Elite ABC Kit, Standard; Vector Laboratories, Inc., Burlingame, CA, USA). The specimens were then rinsed in PBS and reacted with diaminobenzidine (DAB) and H2O2 for 5 min to yield a permanent golden-brown stain.
Besides a series of DAB-stained specimens without counterstaining used for differential interference microscopy (n = 5), which helped distinguish between the tissues layers and vessels31 (link), additional DAB-stained whole mounts (n = 13) were assigned to one or more of several counterstaining conditions to better understand the relationship of the vagal sensory terminals to different tissue elements. Briefly, sets of specimens were counterstained with: (A) Cuprolinic Blue (n = 4; quinolinic phthalocyanine; Polysciences, Inc., Warrington, PA), a pan-neuronal marker that labels enteric neurons32 (link),33 (link), (B) SYTOX Green (n = 10; S7020; Invitrogen), a nuclear stain, to define the smooth muscle cells34 (link) or (C) goat, anti-c-Kit (n = 8; AF1356; R&D Systems, Inc., Minneapolis, MN), an antibody to interstitial cells of Cajal, which are cells involved in smooth muscle contraction.22 (link) The antibody to c-Kit was visualized using either Vector SG peroxidase substrate kit (SK-4700; Vector Lab) or Alexa Fluor 594 (A11058; Invitrogen).35 (link),36 (link)Once stained and counterstained, whole mounts were rinsed in distilled water, mounted serosa-up on gelatin-coated slides, air dried, dehydrated in alcohol, cleared in xylene, and coverslipped with D.P.X. (317616; Sigma-Aldrich, St Louis, MO).
Besides a series of DAB-stained specimens without counterstaining used for differential interference microscopy (n = 5), which helped distinguish between the tissues layers and vessels31 (link), additional DAB-stained whole mounts (n = 13) were assigned to one or more of several counterstaining conditions to better understand the relationship of the vagal sensory terminals to different tissue elements. Briefly, sets of specimens were counterstained with: (A) Cuprolinic Blue (n = 4; quinolinic phthalocyanine; Polysciences, Inc., Warrington, PA), a pan-neuronal marker that labels enteric neurons32 (link),33 (link), (B) SYTOX Green (n = 10; S7020; Invitrogen), a nuclear stain, to define the smooth muscle cells34 (link) or (C) goat, anti-c-Kit (n = 8; AF1356; R&D Systems, Inc., Minneapolis, MN), an antibody to interstitial cells of Cajal, which are cells involved in smooth muscle contraction.22 (link) The antibody to c-Kit was visualized using either Vector SG peroxidase substrate kit (SK-4700; Vector Lab) or Alexa Fluor 594 (A11058; Invitrogen).35 (link),36 (link)Once stained and counterstained, whole mounts were rinsed in distilled water, mounted serosa-up on gelatin-coated slides, air dried, dehydrated in alcohol, cleared in xylene, and coverslipped with D.P.X. (317616; Sigma-Aldrich, St Louis, MO).
