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white adipose tissue of various origins, and liver. First-strand cDNA was prepared from 4 [H9262]g of total RNA using murine reverse transcriptase according to the Pharmacia Biotech procedure. PCR was done using the following primers: f5[H11032]-CCTGTGGCTTTGGTCCTATCTG-3[H11032] and r5[H11032]-CTGC TCAAAGCCACCACCTCTG-3[H11032] (for the ob gene) and f5[H11032]-CGAGAAGATGA CCCAGATCATG-3[H11032] and r5[H11032]-AGTGATCTCCTTCTGCATCCTG-3[H11032] (for the [H9252]-actin gene). After sample denaturation at 94 [H11034]C for 3 min, PCR was done for 40 cycles consisting of denaturation at 94 [H11034]C for 1 min, annealing at 60 [H11034]C for 1 min, and extension at 72 [H11034]C for 2 min. The amplication was terminated by a 10-min nal extension step at 72 [H11034]C. In controls, reverse transcriptase was omitted. Ethidium bromide staining and 1% agarose gel electrophoresis were used to verify the size of the RT-PCR products, that is, 415 bp for the ob gene and 606 bp the [H9252]-actin gene.
Antibodies. Polyclonal antibody 1 was raised in rabbit against the C-terminal fragment [137156] of mouse leptin (Santa Cruz Biotechnology), and polyclonal antibody 2 was raised in rabbit against fragment [138167] of mouse leptin coupled to KLH (keyhole limpet haemocyanin) using EDC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride). The two antibodies gave similar results in western blot analysis and in histological studies. Immunoblots. Unfrozen samples of fundic scrapings and mesenteric adipose tissue were homogenized at 4 [H11034]C in a RIPA buffer containing 0.1 mg ml1
PMSF, 100 [H9262]M benzamidine and 100 mM Na3VO4; they were then solubilized in a boiling Laemmli buffer containing [H9252]-mercaptoethanol. Proteins were separated by 12.5% polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and blotted with polyclonal antibody 1. The immune complexes were revealed by using an enhanced chemiluminescence detection system (Amersham).
Immunohistochemistry. Fed rats were injected intraperitoneally with saline (control) or with CCK-8 (300 [H9262]gkg1) and killed after 15 min. Rat stomachs were removed and xed in Bouins solution. Sections (4 [H9262]m thick) were incubated with antibody 1 diluted to 1 [H9262]gml1 or with antibody 2 diluted 1:50, then with the corresponding biotinylated secondary antibody diluted 1:200, and nally in the avidinbiotin complex diluted 1:100 (Kit ABC Vectastain; Vector Laboratories). Peroxidase activity was revealed by diaminobenzidine. There was no leptin immunostaining in gastric tissues under the following conditions: (1) omission of the primary antibody; and (2) overnight preincubation of the...