Goat anti rat igg

Brains were sectioned in the coronal plane at 30 μm on a sliding knife microtome with a −25 °C freezing stage. Sections were stored in cryoprotectant at −20 °C until further processing. Visualization of antibody-bound sections for immunohistochemistry (IHC) was performed using biotinylated secondary antibodies, avidin-biotin complex (Elite), and 3,3-diaminobenzadine (DAB) substrate kit (Vector Laboratories). The following primary antibodies were utilized: mouse MHC class II I-A^b (BD Pharmingen, 1:2000); Iba-1 (Wako, 1:5000); ICAM-1 (AbD Serotec, 1:2000); rat anti-CD3 (AbD Serotec, 1:4000); hamster anti-CD3 (Santa Cruz Biotechnology Inc., 1:2000); CD11c (BD Pharmingen, 1:500); and CD11b (Invitrogen, 1:1000). Biotinylated secondary antibodies utilized included goat anti-Armenian hamster IgG (Jackson Laboratories, 1:1000); goat anti-mouse F(ab′)2 (Jackson Laboratories, 1:2000); goat anti-rabbit IgG (Vector Laboratories, 1:1000); and goat anti-rat IgG (Vector Laboratories, 1:2000). Sections used for MHC II, CD11c, CD3, and Iba-1 analysis were counterstained with Methyl Green (Vector Laboratories) according to the manufacturer’s protocol. For immunofluorescence, primary antibodies were utilized at concentrations three times the IHC level. Secondary antibodies bound to Alexa (Invitrogen) or DyLight (Jackson Laboratory) fluorophores were utilized at a dilution of 1:500.

Animals were sacrificed at 20–21 weeks of age by perfusion fixation (see supplementary methods). Hypothalamic 30 µm thick coronal sections were selected from each mouse covering the rostral–caudal arcuate (ARC) nucleus region. Immunohistochemical staining of the ionized calcium-binding adaptor molecule 1 (Iba1)- and proopiomelanocortin (POMC)-ir were performed to profile, respectively, the microglial morphology and the neighboring POMC neurons; immunofluorescent co-staining of Iba1 and CD68 was performed to examine microglial phagocytic capacity in the ARC region. See supplementary methods for further information on staining protocols.
Primary antibodies: rabbit anti-iba1 (Ref: 234003, Synaptic Systems), rabbit anti-POMC (Ref: H-029-30, Phoenix Pharmaceuticals), rat anti-CD68 (ab53444, Abcam).
Secondary antibodies: goat anti-rabbit IgG biotinylated (Ref: BA-1000, Vector Laboratories), goat anti-rat IgG, biotinylated (Ref: BA-9400, Vector Laboratories). Fluorescent secondary antibodies: Streptavidin, Alexa Fluor 488 (S32354, Invitrogen), goat anti-rabbit IgG, Alexa Fluor 594 (A11037, Invitrogen).