Ab6721

Five micron paraffin sections from human esophageal tumors were baked overnight at 60 °C, then de paraffinized in 3 changes of xylene and hydrated using graded concentrations of ethanol to deionized water. The tissue sections were subjected to antigen retrieval by 0.01 M sodium citrate buffer (pH 6) in pressure cooker for 5 min (buffer preheated). Following antigen retrieval, all sections were washed gently in deionized water, then transferred in to 0.05 M Tris-based solution in 0.15 M NaCl with 0.1% v/v Triton-X-100, pH 7.6 (TBST). Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min. To reduce further nonspecific background staining, slides were incubated with 5% normal goat serum (Sigma, G9023) for 45 min at RT. All slides then were incubated at 4 °C overnight with anti-RAB14 (Proteintech, 15,662–1-AP, Rabbit polyclonal, 1/100 dilution). After washing with TBST, sections were then incubated with the Goat Anti-Rabbit IgG H&L secondary antibody conjugated with HRP (Abcam ab6721, 1:1000.) Vector Laboratories - ImmPACT DAB Peroxidase (HRP) Substrate Kit (SK4105) was used as the chromogen and hematoxylin (no. 7221, Richard-Allen Scientific, Kalamazoo, MI) as the counterstain. Images were captured at Olympus BX51 upright microscope with Retiga Camera and Bioquant Osteo Image analysis Software.

For immunohistochemistry, representative samples (n = 4, 2 males and 2 females from separate litters) from each group were blocked with 3% hydrogen peroxide and then washed 3 times in phosphate buffered saline and blocked in 1% goat serum or donkey serum with 1% bovine serum albumin. Sections were incubated with the following primary antibodies overnight at 4 degrees: nicotinic acetylcholine receptor α3 (AbCam Cambridge, MA, ab183097, 1:50), α7 (ab10096, 1:200), β2 (ab129276, 1:100), β4 (ab189174, 1:400). Then, sections were washed 3 times in phosphate buffered saline and incubated with HRP conjugated secondary antibody for 1-hour (ab6721, ab6885, 1:250) and diaminobenzidine (DAB) (Vector Laboratories, Bulingame, CA) chromogen was used according to manufacturer’s protocol to identify immunoreactive structures. Coronal suture and whole synchondroses including abutting trabecular bone were digitally isolated for direct comparison between control and nicotine exposed individuals (outlined in Fig. 2d). At least 3 sections 30 μm apart per individual per treatment for each target were analyzed using Image J Software and the IHC Profiler Open Source Plugin for automated scoring of percent positivity^{41 (link)}.