Avidin biotin peroxidase system

Immunohistochemistry was performed as described previously [21] (link). Briefly, an avidin-biotin peroxidase system (Vector Lab, Burlingame, CA, USA) was used to detect anti-insulin receptor rabbit polyclonal antibody (#SC-711: Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Reactions were then visualized with diaminobenzidine (DAB) as substrate (Vector Lab, Burlingame, CA, USA). The sections were counterstained with hematoxylin. All staining procedures were performed as described by the manufacturer.

For histological and immunohistochemical analyses, brains from treated mice were fixed in 4% phosphate-buffered formaldehyde, dehydrated in a STP 120 spin tissue processor (Thermo Scientific) and embedded in paraffin. Sections (5 μm thickness) were cut and mounted onto glass slides, deparaffinized in xylene and rehydrated in a descending series of alcohols, then rinsed in distilled water and stained. Haematoxylin-eosin (HE) staining was carried out according to routine laboratory protocols. For immunohistochemical stainings, enzyme-induced antigen retrieval (for anti-GFAP) using pepsin (1% in 0.01 M HCl) or heat-induced antigen retrieval (all other antibodies) using a 1 × target retrieval solution (Dako) was performed. Primary antibodies used were rabbit anti-GFAP (Dako Z0334, 1:1,000), rabbit anti-Ki67 (CellMarque, Rocklin, CA, 275R-14, 1:200), mouse anti-NeuN (Merck Millipore, Billerica, MA, MAB377, 1:200) and rabbit anti-SFRP1 (Abcam, ab4193, 1:200). Secondary antibodies were diluted 1:500 (anti-mouse, VectorLab, BA-2,000 or anti-rabbit, VectorLab, BA-1,000) and staining was visualized using the avidin-biotin peroxidase system (VectorLab) and freshly prepared diaminobenzidine as chromogen (Dako). Slides were counterstained with haematoxylin, dehydrated and mounted.

Immunohistochemistry was performed as described previously [35] (link). Briefly, an avidin-biotin peroxidase system (Vector Lab, Burlingame, CA, USA) was used to detect two primary antibodies; anti-perilipin rabbit monoclonal antibody (1∶100: Cat. #9349: Cell Signaling Technology, Inc., Danvers, MA) and anti-PPARγ rabbit monoclonal antibody (1∶100: Cat. #2435: Cell Signaling Technology, Inc., Danvers, MA). Reactions were then visualized with diaminobenzidine (DAB) as substrate (Vector Lab, Burlingame, CA, USA). The sections were counterstained with hematoxylin. All staining procedures were followed as instructed by the manufacturer.