Biotinylated anti goat secondary antibody

For F4/80 immunostaining, paraffin sections of visceral fat post HFD were fixed in 10% paraformaldehyde and embedded as described in [29] (link). Sections were deparaffinized and then analyzed with F4/80 macrophage antibody as described previously [29] (link). Briefly, heat induced antigen retrieval was performed with 10 mM Citric Acid (pH 6.0). Sections were blocked with Avidin/Biotin solution (Vector Laboratories kit, #SP-2001) and 10% goat serum (Vector Laboratories kit, #SP-2001). The sections were incubated with primary antibody F4/80 (1∶500, AbD Serotec, #mcA497R) overnight at 4°C, followed by biotinylated anti-goat secondary antibody (1∶200, Vector Laboratories, #BA-9400) for 30 minutes, ABC-AP reagent (Vector Laboratories kit, #AK-5001) for 30 minutes, and vector red alkaline phosphate substrate (Vector Laboratories kit, #AK-5001) for 15 minutes and then counterstained with hematoxylin and eosin (H&E). Liver tissue was similarly fixed and embedded, and then sections were stained with H&E.

The tissue sections were washed twice in 0.1 M phosphate-buffered saline (PBS), twice in 1% triton X-100 (Sigma) for 15 min, and then twice with 0.5% bovine serum albumin (BSA; Sigma) dissolved in PBS for 15 min. The sections were then incubated with goat BMP-2 primary antibody and rabbit IGF-1 primary antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at room temperature in a humidity chamber. After 24 h, the sections were washed twice with 0.5% BSA in PBS and then incubated with either biotinylated anti-goat secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) or biotinylated anti-rabbit secondary antibody (1:200; Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h. After being washed twice with PBS for 15 min, the sections were incubated with an avidin-biotin-peroxidase complex (1:100, Vectastain ABC Kit; Vector Laboratories) for 1 h at room temperature. After another wash with PBS, the sections were stained and reacted with a 0.05% 3, 3-diaminobenzidine (DAB) solution containing hydrogen peroxide in PBS. The reaction was stopped by washing them with PBS and then the slides were dehydrated with solutions of 50, 75, 95, and 100% ethanol and xylene, in that order. The sections were mounted on glass slides with Permount medium solution (Fisher Scientific, Waltham, MA, USA) and micrographs of the sections were taken.