Chicken anti gfp antibody

Dissociated cells were briefly fixed in 4% paraformaldehyde for 15 min at room temperature and washed once with FACS staining buffer (5% fetal bovine serum (FBS) in PBS). Cells were then resuspended in 100 μl of saponin buffer (0.5% saponin and 2% FBS in PBS) and immunostained with anti-CD115-PE cy7 (1:1000, eBioscience #25115282), CD11b-APC-cy7 (1:2000, Biolegend #101226), PU.1 (1:1000, Cell Signaling #2266), chicken anti-GFP antibody (1:1000, Abcam #ab13970), anti-CX3CR1-APC (1:2000, Biolegend #149008), or anti-Ki67-PerCP-efluro710 (1:2000, Invitrogen, #66–5698–82) for 60 min at 4 °C^{57 (link)}.

The cultured seminiferous tubules were fixed with 4% paraformaldehyde in PBS at 4°C overnight. After washing with PBS for 3 min, the tubules were dehydrated in 100% methanol for 30 min at room temperature. The dehydrated tubules were washed with PBS containing 1% Triton-X100 (1% PBST) 4 times for 10 min each, and then blocked with Image-iT™ FX Signal Enhancer (Thermo Fisher Scientific) for 1 h and incubated with primary antibody diluted in blocking buffer at 4°C overnight. After washing with 1% PBST 4 times for 10 min each, the tubules were reacted with secondary antibody diluted in blocking buffer at room temperature for 1 h. After washing with 1% PBST 4 times for 10 min each, the nuclei were counterstained with Hoechst 33342 dye. Specimens were observed with a confocal laser microscope (Olympus FV-1000D). The primary antibodies used were chicken anti-GFP antibody (1:1000; Abcam), rabbit anti-RFP polyclonal antibody (1:1000; MBL), mouse anti-SYCP3 antibody (1:500; Abcam), goat anti-GFRα1 antibody (1:200; R&D Systems), rabbit anti-STRA8 antibody (1:250; Abcam), rabbit anti-γH2AX (1:500; Abcam), and rabbit anti-Mouse HSD3B antibody (1:250; Trans Genic Inc.). The secondary antibodies used were goat anti-chicken IgG, goat anti-rabbit IgG, and goat anti-mouse IgG, conjugated with Alexa 488, Alexa 555 or Alexa 647 (1:200; Invitrogen).

The muscle samples were fixed in a 4% formaldehyde solution at room temperature for 30 min. Subsequently, the samples were rinsed with PBST (PBS with 0.1% Triton X-100) for 4 times, 15 min each time. The samples were then treated with a blocking solution containing 5% BSA in PBS. Following this, the samples were incubated with primary antibodies overnight at 4 °C. Afterwards, the samples were washed again with PBST for 4 times, 15 min each time, and then incubated with secondary antibodies labeled with Alexa Fluor 568 or Alexa Fluor 488 (1:500 dilution, Invitrogen) for 2 h at room temperature. After washing with PBST for 4 times, 15 min each time, the samples were mounted using Slow-Fade mounting medium (Invitrogen) for imaging. The primary antibodies used in this study were chicken anti-GFP antibody (1:1000, Abcam, ab13970); mouse anti-HA antibody (1:1000, Santa Cruz, sc7392); mouse anti-Cnx99A antibody (1:100, DSHB). Representative images were shown for each genotype, with eight individual samples for each genotype analyzed. All tissue immunostaining images were obtained under identical gain settings as well as laser intensities.

Staining of pERK was performed as previously described^{18 (link)} using rabbit monoclonal anti-pERK antibody (4376; Cell Signaling Technology, Beverly, MA, USA) at a 100-fold dilution. Other staining experiments were also performed as previously described^{18 (link)}. The following primary antibodies were used: rabbit anti-CoRest antibody at a 100-fold dilution; rabbit monoclonal anti-myc antibody (2278; Cell Signaling Technology, Beverly, MA, USA) at a 100-fold dilution; mouse nc82 antibody (Developmental Studies Hybridoma Bank, Univ. Iowa, USA) for the detection of the presynaptic protein Bruchpilot at a 50-fold dilution; rabbit anti-DsRed antibody (632496; Takara, Shiga, Japan) at a 200-fold dilution; and chicken anti-GFP antibody (ab13970; Abcam, Cambridge, MA, USA) at a 2,000-fold dilution. The following secondary antibodies were used at 500-fold dilutions: donkey anti-chicken immunoglobulin Y (IgY) Alexa 488 antibody (703-545-155; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA), donkey anti-mouse IgG Alexa Fluor 488 antibody (715-545-150; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA), and donkey anti-rabbit IgG Cy3 antibody (711-166-152; Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA). Images were captured using a confocal microscope LSM780 (Zeiss Microsystems, Jena, Germany) or FV1000 (Olympus, Tokyo, Japan).