Donkey anti goat igg

Cells were fixed with 4% paraformaldehyde (Nacalai Tesque) in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and blocked with 20% Blocking One (Nacalai Tesque) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI; Roche Diagnostics). The following antibodies were used: goat anti-Albumin (ALB; A80-129A, 1:200, Bethyl Laboratories), Rabbit anti-Alpha-fetoprotein (Dako, Glostrup, Denmark), Rabbit anti-MRP2 (JA32-01, Invitrogen), Mouse anti-phospho-Histone H3 (Ser10) Antibody, clone 3H10 (05-806, Millipore), Donkey Anti-Goat IgG (H + L) whole antibody CF 568 Dye (20106, Biotium), Donkey Anti-Goat IgG (H + L) CF657 Dye (20048, Biotium), Donkey Anti-Rabbit IgG (H + L) CF488A (20015, Biotium), and Phalloidin-iFluor™ 488 Conjugate anti-AAT (23115, Bioquest).

Primary antibodies were goat anti-GFP (1:1000, #600-101-215 S, Rockland, Limerick, PA, USA), Rabbit anti-P-p44/42 MAPK (1:1000, #9101 S, Cell Signaling Technology, Frankfurt am Main, Germany), Sheep Anti-Choline Acetyltransferase (1:125, ab18736, Abcam, Berlin, Germany). Secondary antibodies were Donkey anti-Goat IgG conjugated with CF488 (for GFP, 1:1000, #20016-1, Biotium), Donkey anti-Rabbit IgG conjugated with CF594 (for pERK, 1:1000 for the OB and 1:500 for the HDB, #20152, Biotium), biotinylated Donkey anti-Sheep IgG (for ChAT, 1:250, #20191-1, Biotium). All antibodies were diluted in the blocking solution.
All purchased antibodies were validated for target and species specificity as indicated in the data sheets of the respective companies’ websites.

Animals were deeply anesthetized with sodium pentobarbital, and were exsanguinated by intracardiac perfusion with phosphate buffered saline (PBS) followed by fixation with perfusion of 4% paraformaldehyde (PFA). Whole brain was post-fixed in 4% PFA for 1 h at 4°C and cryoprotected. Each sample embedded in O.C.T. mounting medium (Sakura Finetek) was cut into 12-μm-thick sections at around −20°C and stored on slide glasses at −80°C. Sections were incubated at 4°C over night with primary antibodies: rabbit anti-Ki-67 (1:500, Abcam), goat anti-doublecortin (DCX) (1:1,000, Santa Cruz), rabbit anti-calretinin (1:1,500, SWANT) rabbit anti-calbindin D-28 K (1:400, SWANT), and mouse anti-Parvalbumin (1:7,500, SWANT). Then, the sections were incubated at room temperature for 2 h with fluorescence-conjugated secondary antibodies (donkey anti-goat IgG (H + L, 1:1,000, Biotium), goat anti-rabbit IgG (H + L, 1:1,000, Biotium) or goat anti-mouse IgG1 (1:2,000, Biotium). Signals were detected with a fluorescence microscope (AxioImager M1, Zeiss).