Mouse anti gfp antibody

Whole-mount antibody staining was performed using a rabbit anti phospho-histone H3 (pH3) primary antibody (Cell Signaling Technology) at 1∶100 dilution. Mouse anti-GFP antibody (1∶1000) (Proteintech) was used to detect GFP in Ptf1a: GFP transgenic line. A Cy3 labeled goat anti-rabbit second antibody (1∶200) (Proteintech) and an Alexa Fluor 488 conjugated goat anti-mouse second antibody (1∶200) (Invitrogen) were used as secondary antibodies. For TUNEL assay, transverse sections were prepared using a Leica VT1000S vibratome at 200 µm intercals. Then the staining was performed using In Situ Cell Death Detection Kit (Roche) according to the manufacture’s instructions. The images were then observed under a Zeiss LSM 510 meta confocal microscope (Zeiss, Oberkochen, Germany).

For the Western blot analysis, the total protein was extracted with protein extraction kits (Solarbio, Beijing, China) following the manufacturer’s instructions. Protein was separated by 10% SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) using a transfer buffer at 64 V for 2 h. The procedure of Western blot analysis was described previously [47 (link)]. The membranes were washed three times with Tris-buffered saline (TBS, 5 min each) and then blocked with 5% nonfat dry milk in TBST (TBS with 0.1% Tween 20) for 1 h at 25 °C with 50 rpm shaking. Then, the membranes were incubated with primary antibody (mouse anti-GFP antibody (1:10,000, (Proteintech, IL, USA)), mouse anti-Bax antibody (1:10,000, Proteintech)) at 4 °C overnight. After washing three for 5 min in TBST, membranes were then incubated with goat anti-mouse IgG (H+L) antibody (Proteintech) as secondary antibody at 1:10,000 dilutions for 2 h, followed by three washes at room temperature. The Protein bands were detected with chemiluminescence horseradish peroxidase substrate (Merck Millipore, Burlington, MA, USA) and photographed following the manufacturer’s instructions.

Protein samples were separated on SDS-PAGE gels and then electroblotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h in 5% non-fat milk in TBST (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.2% Tween 20) followed by incubation with primary antibodies: mouse anti-GFP antibody (Cat. 66002-1-Ig, Proteintech, Rosemont, IL, United States), rabbit anti-GFP antibody (Cat. ab290, abcam, Cambridge, United Kingdom), mouse anti-FLAG antibody (Cat. F3165, Sigma), rabbit anti-FLAG antibody (Cat. 20543-1-AP, Proteintech), rabbit anti-Myc antibody (Cat. 16286-1-AP, Proteintech), mouse anti-β-Actin antibody (Cat. 66009-1-Ig, Proteintech), rabbit anti-CDK2 antibody (Cat. 10122-1-AP, Proteintech), or rabbit anti-pCDK2 antibody (Cat. ab194868, abcam). Two secondary antibodies were used: goat anti-mouse antibody (Cat. ab216776, IRDye 680RD, abcam) and goat anti-rabbit antibody (Cat. ab216773, IRDye 800RD, abcam). Signals were captured by an Amersham Typhoon (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by ImageQuant TL (GE Healthcare).