Donkey anti rabbit alexa 405

Free-floating sections were washed three times in PBS, followed by 1 hour blocking in PBS with 10% donkey serum. Sections were incubated in PBS with 10% donkey serum and primary antibodies for 48 hours at 4 °C: rabbit anti-TMEM119 (1:400, Abcam, ab209064), rat anti-CD68 (1:200, Bio-Rad, MCA1957GA), rabbit anti-Iba1 (1:1000, Wako, 019–19741), guinea pig anti-Perilipin 3 (1:200, Progen, G37). After the primary antibody incubation, sections were washed three times in PBS and incubated in PBS with 10% donkey serum and secondary antibodies for 3 hours at room temperature (RT): donkey anti-rabbit Alexa 555, donkey anti-rabbit Alexa 647, donkey anti-rabbit Alexa 405 (all 1:500, Invitrogen), donkey anti-rat Cy5, donkey anti-guinea pig Alexa 488 (all 1:500, Jackson Immuno Research). Sections were washed once in PBS and incubated in PBS with BODIPY™ 493/503 (1:1000 from 1 mg/ml stock solution in DMSO; ThermoFisher) to stain lipid droplets and Hoechst 33342 (1:2000, ThermoFisher) for nuclear counterstaining for 15 min at RT. Sections were mounted on microscope slides and embedded with Vectashield (H-1000, Vector Laboratories). Note that for successful lipid droplet staining, antigen retrieval steps and treatment with detergents have to be avoided, and sections should be embedded while still wet.