Immunohistochemical and fluorescent staining and microscopy protocol (Stengel et al. )

Three ad libitum fed rats were euthanized by CO₂ anesthesia followed by cervical dislocation. Esophagus, stomach, small intestine, colon, ancreas, liver, spleen, heart, gastrocnemius muscle, lung, kidney, adrenal gland, testis, visceral fat, subcutaneous fat, and pituitary land are removed and ex situ fixed in Bouin’s fixative for 2 h. The tissues were rinsed in 50% ethanol three times to remove fixative residues and processed following standard procedures until paraffin block embedding. Five um sections were cut using a microtome, deparaffinized and re-hydrated with graded xylene-alcohol series and washed with phosphate buffered saline (PBS) three times for 15 min before immunostaining. Endogenous peroxidase was inactivated by 0.3 % hydrogen peroxide in PBS for 30 min and unspecific binding was reduced by pre-treatment with 3 % normal goat serum. Sections were incubated overnight at 4 ºC in anti-rat nesfatin-1 (1-82) polyclonal antibody at a titer of 1:2,000 (Catalog #: H-003-22. Phoenix Pharmaceuticals, Burlingame, CA) in 0.3% Triton X-100 in PBS, then in biotinylated goat anti-rabbit IgG (1:1,000, Jackson ImmunoResearch, NJ) in 0.3% Triton X-100 in PBS for 1 h at room temperature followed by incubation with avidin-biotinperoxidase complex in 0.3% Triton X-100 in PBS (ABC, Vector, Vermont, CA) for 1 h at room temperature. Sections were developed with 3,3’-diaminobenzidine tetrahydrochloride hydrate and hydrogen peroxide (Sigma Chemical Co, St. Louis, MO) for 10 min and frequently checked on the light microscope. Each step of incubation was followed by a 3 x 5 min washing step in PBS. Sections were mounted on Fisher SuperFrost Plus slides, air-dried for 24 h and completely dehydrated through a gradient of ethanol and xylene. Slides were studied using light microscopy.

The same protocol was applied for immunostaining after pre-absorption of the antinesfatin-1 antibody. Fifty μg rat nesfatin-1 (Phoenix Pharmaceuticals, Burlingame, CA) were incubated with 1 ml rabbit anti-rat nesfatin- 1 (1:2,000) in 0.3% Triton X-100 in PBS for 2 h at room temperature followed by 22 h at 4 ºC. The solution was centrifuged for 15 min at 16,000 g and the supernatant was used for staining as described above.

For immunofluorescent double-labeling the gastric corpus was sectioned. After pre-treatment with normal goat serum as described above, sections were incubated overnight at 4 ºC in antinesfatin-1 (1-82) antibody (1:500) (Catalog #: H-003-22) together with either guinea-pig anti-histidine decarboxylase (HDC, 1:1,000, Eurodiagnostics, Sweden), mouse anti-ghrelin serum D4-7.1 (1:2,000, Eli Lilly Research Laboratories, Indianapolis, IN) or mouse anti-somatostatin (1:500, SST CURE S6, UCLA Digestive Disease Research Center, CA) antibodies in 0.3% Triton X-100 in PBS. Tetramethyl rhodamine iso-thiocyanate (TRITC) conjugated goat anti-rabbit IgG (1:2,000, Jackson ImmunoResearch Laboratories, West Grove, PA) together with either fluorescein isothiocyanate (FITC)-labeled anti-mouse (1:500, Jackson ImmunoResearch Laboratories) or FITC-labeled anti-guinea-pig (1:100, Jackson ImmunoResearch Laboratories) antibodies in 0.3% Triton X-100 in PBS were added for 2 h at room temperature. Each step of incubation was followed by a 3 x 5 min washing in PBS. Counterstaining was performed using 4’,6-diamidino-2-phenylindole (DAPI, Sigma Chemical Co, St. Louis, MO) 1:1,000 for 6 min. The slides were mounted with antifade mounting medium (Vector Laboratory Inc., Burlingame, CA, USA) and visualized by confocal microscopy (Zeiss, LSM 510, Germany).