Hrp conjugated secondary goat anti rabbit igg

Anti-GFP antibody staining largely followed the methods described by Stern (49 (link)) and Streit and Stern (50 (link)). Embryos were processed for anti-GFP antibody staining either immediately after collection (and fixing in 4% PFA overnight at 4 °C or at room temperature for 20 min) or following in situ hybridization. Embryos stained either with rabbit anti-GFP primary (Life Technologies, 1:2,000 dilution) followed by an HRP-conjugated secondary goat anti-rabbit IgG (Santa Cruz, 1:2,000 dilution) or with a goat anti-GFP primary antibody (Rockland, #600–101-215; 1:500 dilution) followed by donkey anti-goat Alexa Fluor 488 secondary antibody (1:1,000 dilution). HRP was revealed using DAB (Sigma, #D5637). pH3 was detected using mouse anti-histone H3, phospho S10 IgG1 (Abcam-ab443110, 1:500 dilution) followed by a donkey anti-mouse IgG Alexa Fluor 647 (Invitrogen, #A31571, 1:1,000 dilution).

The harvested cells were suspended in cell lysis buffer. After 12% SDS-PAGE, the proteins were transferred onto a PVDF membrane (Millipore, Temecula, CA, USA) and blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk for 1 hr. The membrane was incubated with the rabbit anti-KITLG (1:500; Abcam, Cambridge, UK) or rabbit anti-c-KIT (1:1000; Cell Signaling Technology) primary antibodies at 4°C overnight. Then, the membrane was incubated with HRP-conjugated secondary goat anti-rabbit IgG (1:2000; Santa Cruz) for 1 hr. The proteins were detected by using ECL chemiluminescence. A mouse anti-GAPDH (1:1000; Applygen, Beijing, China) antibody was used as an internal control.

The whole-cell lysates from SH-SY5Y cells (30 μg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in an 8–12% gel, and the proteins were transferred to a nitrocellulose membrane. After the blots were washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween 20), the membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with the appropriate primary antibodies: against p-AMPK, PGC-1α, BCL2, BAX, cleaved-caspase-3, cleaved-PARP-1, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed, and the primary antibodies were detected using HRP-conjugated secondary goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).