Fitc labeled goat anti rabbit secondary antibody

Rats were anaesthetized with isoflurane (4% induction and 1.5–2% maintenance) and transcardially perfused with 200 ml of saline containing heparin (50 i.u./l), followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2). The brains were dissected out and post-fixed by immersion for 1 h at RT in the same fixative. Later, brains were cryoprotected with a 30% sucrose solution in 0.1 M PB at – 20 °C and brain sections of 6 μm thickness were obtained using a freezing-sliding microtome. Sections were washed in a PBS solution (0.1 M, pH 7.4) and then stored at 4 °C in a freezing solution (30% glycerol and 30% ethylene glycol in 0.1 M PB at pH 7.4). Fixed brain sections were rinsed in PBS with 0.2% TritonX-100, and then blocked for 2 h with 1% BSA and 5% normal serum in PBS-Tx. After incubated overnight at 4 °C with 1% BSA, 2% normal serum and primary rabbit anti-POMC antibodies, and subsequently reacted with FITC-labeled Goat Anti-Rabbit secondary antibody (Invitrogen). The nucleus was stained with DAPI (4,6-diamidino-2-phenylindole). Images were captured using a FW1000 confocal microscope.

Another set of rats in experiment 2 (5–6 per group) were anaesthetized and transcardially perfused with 200 ml of saline containing heparin (50 i.u./L), followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2). Each rat’s brain was removed, post-fixed in 4% paraformaldehyde for 1 h, placed in phosphate-buffered saline containing 30% sucrose and stored at 4 °C. Brain sections of 6 μm thickness were made using a cryostat at −20 °C. Fixed brain sections were blocked with serum of the appropriate species, penetrated with 0.2% TritonX-100, and treated with primary rabbit anti-POMC antibodies, and subsequently reacted with FITC-labeled Goat Anti-Rabbit secondary antibody (Invitrogen). The nucleus was stained by DAPI (4,6-diamidino-2-phenylindole). Images were captured under a FW1000 confocal microscope.