Rabbit anti iba1

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).

Twenty μm coronal sections were cut at −20 °C in a cryostat, rinsed in 0.1 M PBS and blocked in 0.1 M PBS with 10% normal goat serum and 0.3% Triton X-100. Sections were then incubated overnight at 4 °C with primary antibodies dissolved in blocking solution: rabbit anti-mCherry (Thermo Fisher, 1:100); rabbit anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA, 1:100); rabbit anti-nitric oxide synthase-2 (Santa Cruz, Dallas, TX, USA, 1:100); mouse anti-CD45 (Santa Cruz, 1:100); rabbit anti-Iba1 (Synaptic Systems, Gottingen, Germany, 1:200). The next day, sections were incubated with secondary antibodies for 2 h at room temperature, as follows: Alexa Fluor 488 goat anti-rabbit (Molecular Probes, Eugene, OR, USA), 1:1,000; Alexa Fluor 488 goat anti-mouse (Molecular Probes), 1:1,000. In addition, 10 μM DAPI (Molecular Probes) was added to the secondary antibody solution in order to label cell nuclei.