Rat anti gfp

The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Delta, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-pS/TQ (Cell Signaling Technologies, Danvers, MA, USA), 1:1000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma-Aldrich), 1:1000; rabbit anti-β-gal (Upstate Biotechnology Inc., Lake Placid, NY, USA), 1:1000; and anti-CCleaved caspase-3 (Cell Signaling Technologies), 1:1000; rabbit anti-pJNK antibody (Cell Signaling Technologies). The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor^® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.

The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Dl, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma–Aldrich), 1:1000; rabbit anti-H3K9me3 (Millipore, Billerica, MA, USA), 1:200; and, mouse anti-HP1 (DSHB, Iowa City, IA, USA), 1:200. The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor^® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.

Cultured cells were fixed with 4% paraformaldehyde (PFA) in 0.01M PBS at room temperature or methanol/acetone (1:1) at −30°C for 15 min, and then rinsed in PBS. For F-actin staining, transfected cells were fixed with 4% PFA, followed by permeabilization with 0.5% TritonX-100 in PBS for 10 min. After blocking with 10% normal goat serum and 0.1% TritonX-100 in PBS, cells were reacted with primary antibodies overnight at 4°C. After washing, cells were labeled with species-specific secondary antibodies conjugated to Alexa Fluor 488 or 594 (1:1000, Life Technologies) and counterstained with Hoechst 33342 (Sigma) or DRAQ5 (Abcam). Primary antibodies used were: rat anti-GFP (1:2000, Nacalai Tesque), rabbit anti-Dpy19L1 (C-ter, 1:100, Abgent; N-ter, 1:250, Abcam), chicken anti-Calreticulin (1:1000, Abcam), mouse anti-Calreticulin (1:200, Abcam), rabbit anti-active Caspase-3 (1:1000, BD Biosciences), Phalloidin (CF dye conjugates, 1:200, Biotium) and mouse anti-α-Tubulin (1:2000, Sigma). Pictures were taken with a digital camera (DP72, Olympus). Confocal images were captured with a confocal laser scanning microscope (FV1200). Intensity profile analysis was performed using FV10-ASW software (Olympus). For colocalization analysis, the scatter plots of red and green pixel intensities of confocal images were created using Image J/Fiji software.

Embryos were stained with antibodies as previously described [37 (link)] and observed with a Zeiss LSM 700 or LSM 810 confocal laser microscope, and the results were analyzed using the LSM image browser (Zeiss, Jena, Germany) and ImageJ software (Version 13.0.6, NIH, Bethesda, MD, USA) [38 (link)]. We used the following primary antibodies: rat anti-Elav (7E8A10, 1:500) [39 (link)], mouse anti-NICD (C17.9C6, 1:250) [40 (link)], mouse anti-Crumbs (Cq4, 1:250) [41 (link)], rat anti-E-Cadherin (DCAD2 1:500) [42 (link)], guinea pig anti-FL-Hrs (GP30, 1:1000) [43 (link)], rabbit anti-Rab7 (1:5000) [44 (link)], rabbit anti-Rab11 (1:4000) [44 (link)], rabbit anti-GM130 (1:50, Abcam, Cambridge, MA, USA) [45 (link)], rabbit anti-GFP (1:250, 598 MBL) [46 (link)], and rat anti-GFP (1:250, Nacalai Tesque, Kyoto, Japan).
We used the following secondary antibodies: Cy3-conjugated donkey anti-mouse, Cy5-conjugated anti-rabbit, Cy-5 conjugated anti-rat, Cy5-conjugated anti-guinea pig, Alexa488-conjugated donkey anti-rat, and Alexa488-conjugated donkey anti-rabbit (all from Jackson Immunoresearch, West Grove, PA, USA).

Mice were anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan) and transcardially perfused with 4% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) in phosphate-buffered saline. Coronal brain sections (100 µm thick) prepared with a vibratome-type tissue slicer (DTK-1000, Dosaka-EM, Kyoto, Japan) were immunostained with rabbit anti-GFAP (1:500, DAKO, Glostrop, Denmark) and rat anti-GFP (1:1000, Nacalai Tesque) antibodies as primary antibodies, and Alexa Fluor 555 Goat anti-rabbit (1:200, Molecular Probes, Eugene, OR) and Alexa Fluor 488 Goat anti-rat (1:200, Molecular Probes) antibodies as secondary antibodies. The stained sections were observed with a confocal laser scanning microscope (FV-300, Olympus, Tokyo, Japan) equipped with objectives (4×, NA 0.16 and 20×, NA 0.75, Olympus).