Sheep anti αmsh antibody

In all, 10- to 12-week-old mice (n = 7–8 per group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-µm thick, and processed for immunofluorescence using standard procedures^{7 (link)}. The primary antibodies used for IHC were as follows: sheep anti-αMSH antibody (1:40,000, Millipore, cat #AB5087), rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat #H003-53), rabbit anti-LC3B (1:500, Cell Signaling, cat #2775), rabbit anti-p62 (1:1,000, Abcam, cat #ab64134), rabbit anti-GFP (1:1,000, Invitrogen, cat #A-6455), guinea pig anti-insulin (1:500, Abcam, cat #ab7842), and rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems, cat #139103). The primary antibodies were visualized with Alexa Fluor 568 donkey anti-sheep IgG (1:200, Invitrogen, cat #A21099), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A21206), Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen, cat #A21202), Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A10042), or Alexa Fluor 488 goat anti-guinea pig IgG (1:200, Invitrogen, cat #A11073). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat #H3569) to visualize cell nuclei.

15-17-weeks old male mice (n = 4/group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-um thick, and processed for immunofluorescence using standard procedures (Bouret et al., 2004 (link)). Briefly, sections were incubated with a sheep anti-αMSH antibody (1:40,000, Millipore). The primary antibody was visualized with Alexa Fluor 488 donkey anti-sheep IgG (1:200, Millipore). Sections were counterstained using bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei. Two sections through various anatomical compartments of the PVH (PVHmpd and PVHpml, neuroendocrine; and postPVH, autonomic) were acquired using a Zeiss LSM 710 confocal system equipped with a 20X objective. For the quantitative analysis of fibers density, each image plane was binarized to isolate labeled fibers from the background and to compensate for differences in fluorescence intensity. The integrated intensity, which reflects the total number of pixels in the binarized image, was then calculated for each image. This procedure was conducted for each image stack.

Ten- to twelve-week-old mice were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-μm thickness, and processed for immunofluorescence using standard procedures [8 (link),37 ]. The primary antibodies used for IHC were as follows: rabbit anti-POMC (1:20,000, Phoenix Pharmaceuticals, cat# H029-30), sheep anti-αMSH antibody (1:40,000, Millipore, cat# AB5087), and rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat# H003-53). The primary antibodies were visualized with Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Millipore, cat# A21206), Alexa Fluor 568 donkey anti-sheep IgG (1:200, Millipore, cat# A21099), or Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Millipore, cat# A10042). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat# H3569) to visualize cell nuclei.