Biotin sp affinipure goat anti rabbit igg

Adipose tissue IHC staining was performed as described before (Bal et al, 2017). Mouse adipose tissues (brown and white adipose tissue) were fixed with 10% formalin, embedded with paraffin, and sliced into 10‐μm sections. The sections were mounted on slides and dehydrated with sequential alcohol gradient (0, 50, 70, 80, 90 and 100%). Antigen retrieval was performed by incubating with EDTA reagent at 90°C for 10 min. The sections were then incubated with 0.3% H₂O₂ for 30 min, followed by blocking solution buffer for 1 h at room temperature. Subsequently, the sections were incubated in rabbit anti‐phospho‐HSL (1:1,000) or rabbit anti‐UCP1 (1:1,000) overnight, followed by Biotin‐SP‐AffiniPure Goat Anti‐Rabbit IgG (111‐065‐003; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The sections were then visualized by incubation with the ABC kit (PK‐4000; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Sections were then treated with diaminobenzidine (D12384; Sigma) for 5 min, followed by dehydration in a graded ethanol series from 50 to 100% and a final wash in xylene. Images were obtained using Upright microscopes, and Image‐Pro Plus software was used to quantify grayscale. Up to six fields of view were captured from the same location within each adipose tissue.

Immunohistochemical staining for DICER1 was performed using 1:200 polyclonal rabbit anti-DICER1 primary antibody (Sigma-Aldrich HPA000694), and amplification with 1:1,000 Biotin-SP AffiniPure Goat Anti-rabbit IgG and 1:500 peroxidase streptavidin (Jackson ImmunoResearch Laboratories 111-065-144 and 016-030-084, respectively). Samples were developed using ImmPACT DAB peroxidase (HRP) substrate (Vector SK-4105). The rest of the protocol has been detailed elsewhere (19 (link)). Images were acquired using a Keyence BZ-X710 microscope and processed with the BZ-X Analyzer software (Keyence).

HeLa cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, #08458-45) containing 10% fetal bovine serum (Equitech Bio, #268-1). Rabbit polyclonal anti-TOM20 antibody was purchased from Santa Cruz Biotechnology (#sc-11415), and mouse monoclonal anti-GM130 antibody was from BD Biosciences (#610822). Alexa Fluor 555 conjugated anti-mouse IgG (#A31570), Alexa Fluor 555 conjugated anti-rabbit IgG (#A31572), Alexa Fluor 555 conjugated streptavidin (#S21381), Alexa Fluor 647 conjugated streptavidin (#S21374), and Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (#W32466) were from Thermo Fisher Scientific. Biotinylated WGA (#B-1025) was from Vector Laboratories. DyLight 549 (#S000-42) and DyLight 649 (#S000-43) conjugated streptavidins were from Rockland. HiLyte Fluor 555 (#AS-60666) and HiLyte Fluor 647 (#AS-60667) conjugated streptavidins were from Anaspec. iFluor 555 (#16989) and iFluor 647 (#16996) conjugated streptavidins were from AAT Bioquest. ATTO 647 (#AD647-61) streptavidin was from ATTO-TEC. SPICA dye-conjugated streptavidin was from Fujifilm Wako Chemicals. Biotin-SP AffiniPure goat anti-rabbit IgG (#111-065-144) was purchased from Jackson ImmunoResearch.