Donkey anti mouse igg h l

Cells were directly grown on glass coverslips (0.17 mm thickness, 1.5 H high performance; Marienfeld Superior) placed in 24-well plates. Cells were fixed with 4% paraformaldehyde (20 min; RT), permeabilized with methanol (10 min RT) and blocked with 5% bovine serum albumin (5% BSA diluted in phosphate-buffered saline [PBS]-Triton 0.1%). Primary antibodies of interest were incubated overnight at 4°C. Secondary antibodies conjugated to Alexa fluorophores were incubated 1 hr RT in the dark. The following conjugated secondary antibodies (Invitrogen) were used: goat anti-rabbit IgG H+L (Alexa-488 or -647); donkey anti-mouse IgG H+L (Alexa-555, -561, -633, or -647). After incubation, samples were washed with PBS-T (x3) and nuclei were stained with Hoechst (25 µg/ml diluted in 5% BSA-PBS-T; 1 hr RT in the dark). Five PBS-T washes and a final MiliQ water were performed. Coverslips were mounted using Pro-long glass (Invitrogen). Preparations were maintained 24–48 hr in the dark at RT and then stored at 4°C up to image acquisition.

HL7702 cells were plated into 24-well plates and grown to 50% confluence. Following plasmid transfection and H₂O₂ induction, immunofluorescence was performed. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100 for 15 min. Cells were then blocked with 10% donkey serum for 60 min, incubated with primary antibodies overnight at 4 °C, and immunostained with secondary antibodies for 60 min at 23–25 °C. Primary antibodies specific for ASC (#ab180799; Abcam, Cambridge, MA, USA) and HBx (#ab235; Abcam, Cambridge, MA, USA) were used. The secondary donkey anti-mouse IgG (H + L), Alexa Fluor 488 (#A-21202; Invitrogen, Carlsbad, CA, USA), donkey anti-rabbit IgG (H + L), and Alexa Fluor 555 (#A-31572; Invitrogen, Carlsbad, CA, USA) antibodies were used. Finally, cells were observed using fluorescence confocal microscopy (Zeiss FM780, Jena, Germany). Pictures were taken under 63 × oil microscope and excitation light wavelengths of 405 nm, 488 nm, and 594 nm were selected for layer scanning with a scanning interval of 0.35 um/layer. The fluorescence excitation displayed blue, green, and red, respectively.

Cells were collected at d0, d4, d6, d7, d8, d10 and d14 from in vitro neural differentiation and fixed with 70% ice cold ethanol for 20 min at RT. Next cells were washed with washing solution (PBS with 0.5% BSA). For denaturation 2 M HCl was used for 10 min at RT. After washing the neutralization of the residual acid was done with 0.1 M sodium borate (pH 8.5). Cells were washed and single or double stained with mouse monoclonal PAX6 (Hybridoma Bank, Iowa, USA) and rabbit polyclonal RYBP/DEDAF (Merck-Millipore, Cat.No AB3637) primary antibodies for 30 min at RT followed with Alexa Fluor 647 labelled Donkey anti Mouse IgG (H + L) (Invitrogen, Cat.No A-31571) and Alexa Fluor 488 labelled Donkey anti Rabbit IgG (H + L) (Invitrogen, Cat.No A-21206), respectively for 20 min at RT. All antibodies were diluted in dilution solution (washing solution containing 0.5% BSA and 0.5% TWEEN-20 (Sigma-Aldrich, Cat.No: P5927) according to the manufacturers. For negative controls, first antibodies were omitted and cells were labelled only with secondary antibodies at each examined time points.
We measured the fluorescence of 50,000 cells/sample with Becton Dickinson FACS Calibur fluorescent flow cytometer with 488 nm and 633 nm laser for Alexa Fluor 488 and Alexa Fluor 647 labelled secondary antibodies, respectively. Data were analyzed by BD CellQuest Pro Version 6.0 software.

The following materials are used in the cell culture: L-leu (#L8912), lysine (Lys, #L9037), arginine (Arg, #A6969), Collagen Ⅰ (#SCR103), Basic Fibroblast Growth Factor (bFGF, #13256-029), BSA (A1933), and DMSO (#C6295), which were purchased from Sigma-Aldrich, USA. DMEM (#11995065), L-leu-deprived DMEM powder (#88425), fetal bovine serum (FBS, #10099141C), Antibiotics-antimycotic (#15240062), and Dulbecco phosphate-buffered saline (DPBS, #14190144) were purchased from Gibco, Life, Technologies, Grand Island, NY, USA. Culture dish (10 and 6 cm), Cell ware 6, 24, and 96-well plates, Matrigel and cell strainer, were purchased from Corning, NY, USA. Accutase, Cell Detachment Solution was purchased from innovative cell technologies, SD, USA. Mouse monoclonal anti-PAX7 (#sc-81648) was purchased from Santa Cruz Biotechnology, CA, USA. Donkey anti-Mouse IgG (H + L) was purchased from Invitrogen (#A10036, Invitrogen, Carlsbad, CA, USA). DAPI (#C0065), Triton X-100 (#9002-93-1), and Tween 20 (#9008-64-5) were purchased from Solarbio (Beijing, China). CCK 8 was purchased from Good Laboratory Practice Bioscience, Montclair, CA, USA.

Immunostaining was performed at the Biopathology Institute. Co., Ltd. (Kunisaki, Oita, Japan). After 4% paraformaldehyde fixation, the oral mucosa around the mandibular molars was permeabilized and blocked using Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan). The slides were incubated with antibodies to p21 (ab188224, Abcam, Cambridge, UK) γH2AX (Sigma Aldrich, St. Louis, MO, USA), phospho-histone H2A.X (Ser139) (clone JBW30, 05-636; MerckMillipore, Waltham, MA, USA), and NF-κB p65 (200-301-065, Rockland Immunochemicals, Pottstown, PA, USA) at 4 °C overnight. After incubation, the sections were washed with phosphate buffer saline (PBS).
The Alexa Fluor 488-conjugated secondary antibodies were: chicken anti-rabbit IgG (H + L) (A21441; Invitrogen, Carlsbad, CA, USA), donkey anti-rat IgG (H + L) (A21208; Invitrogen), and donkey anti-mouse IgG (H + L) (A21202; Invitrogen), and sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, SJ217; Dojindo, Kumamoto, Japan). We observed the sections with a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Images are representative of at least three independent experiments. The numbers of positive cells in the perigingival region were counted using ImageJ v1.52a. Three individuals performed a quantitative analysis of each immunolabeled area, and their scores were averaged.

Day 4 hPGCLC aggregates were fixed in 4% PFA for 20 min at room temperature. After PBS washes and incubation in 30% sucrose, aggregates were embedded in OCT (VWR, 361603E) and sectioned at a thickness of 10 μm. Samples were permeabilized for 20 min in PBS/0.2% Triton X100 (PBST) and blocked for 1 h in blocking solution (Santa Cruz, sc-516214). Samples were then incubated with the following primary antibodies diluted in blocking buffer at room temperature for 2 h: anti-OCT4 (1:200, catalog no. ab181557; Abcam); anti-TFAP2C (1:100, catalog no. sc-12762; Santa Cruz); anti-SOX17 (1:200, catalog no. AF1924; R&D). After PBS washes, samples were incubated with the following secondary antibodies diluted 1:1000 in PBS: Donkey anti-rabbit IgG (Alexa Fluor 594, catalog no. A21207; Invitrogen); Donkey Anti-Goat IgG H&L (Alexa Fluor 488, catalog no. ab150129; Abcam); Donkey anti-Mouse IgG (H&L) (Alexa fluor 594, catalog no. R37115; Life Technologies). Sections were then washed with PBS and incubated for 10 min with DAPI. A coverslip was then placed on the slides in Vectashield vibrance (Vector Labs, H-1800-10). Sections were imaged using the SP5 inverted confocal microscope (Leica).