Can get signal immunostain

The undifferentiated SH-SY5Y cells transiently and stably expressing α-synuclein and the differentiated SH-SY5Y cells by retinoic acid and BDNF were treated with SCH28080, vonoprazan, and bafilomycin A1 for 48 h. The cells were fixed with ice-cold methanol for 5 min, washed with Tris-buffered saline (TBS), and permeabilized with permeabilization buffer containing 0.3% Triton X-100 and 0.1% BSA in TBS for 15 min at room temperature. Non-specific binding of the antibody was blocked with 2% BSA in TBS, and the cells were incubated with an anti-phosphorylated α-synuclein antibody (pSyn#64) (1:100) in Can Get Signal immunostain (Toyobo) for 15 h at 4 °C. The cells were washed with TBS and incubated with the Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:100) for 1 h at room temperature. Immunofluorescence images were visualized by using a Zeiss LSM 700 laser scanning confocal microscope. The area (dimension) of the fluorescent signal due to phosphorylated α-synuclein was measured by Zen3.3 software (Zeiss).

To immunochemically detect CoQ biosynthetic proteins, rabbit polyclonal antisera were prepared by Sigma–Aldrich by injecting rabbits with specific peptides of Coq proteins (53 (link)). The specificity of antisera against each of the CoQ biosynthetic proteins (Dlp1, diluted 1:1000; Coq4, diluted 1:500) was assessed by Western blot analysis. Preparation of cell lysates and detection of CoQ biosynthetic proteins by immunoblotting S. pombe cell lysates were performed as described previously (28 ). S. pombe WT (PR110) cells were inoculated into 55 ml YES main cultures with or without Bz (initial cell density ∼1 × 10⁵ cells/ml) and incubated with rotation at 30 °C for 2 days and then harvested. Lysate proteins were separated by SDS-PAGE, after which Western blot analysis was performed using an ECL detection system (GE Healthcare) or Amersham ECL Western Blotting Detection Reagent RPN2106 (Cytiva). Rabbit polyclonal antibodies against the PSTAIRE peptide (Cdc2, diluted 1:1000) were purchased from Santa Cruz Biotechnology. Horseradish peroxidase–conjugated anti-rabbit immunoglobulin G antibody (Promega) was used as secondary antibody (diluted 1:2000). These antibodies were dissolved in Can Get Signal immunostain (TOYOBO), an immunoreaction enhancer solution. For quantification of protein bands, ImageJ (https://imagej.nih.gov/ij/download.html) was used.

Immunostaining was carried out as described previously [30 (link)]. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by permeabilization with 0.1% Triton X-100 in PBS. Cells were stained with primary antibody diluted with Can-Get-Signal immunostain (Toyobo) and secondary antibody diluted with 2% FCS in PBS. Fluorescence images were obtained using a DMI6000B fluorescence microscope (Leica Microsystems) equipped with a PL Apo 63x oil objective lens and CCD camera (Cool SNAP HQ, Roper Scientific).

Embryonic stage was determined as E11.5 on the basis of the number of somites, while the sex of the embryos was determined by polymerase chain reaction (PCR) for the Sry gene using the following primer pairs: Sry-F: 5′-ggttgcaatcataattcttcc-3′ and Sry-R: 5′-cactcctctgtgacactttag-3′.
For section immunostaining, embryonic gonads were fixed with 4% paraformaldehyde overnight at 4°C and then embedded in paraffin. Whole gonads were sectioned (6 μm) and autoclaved with Antigen Unmasking Solution (Vector Laboratories, Inc., USA). After the samples were subjected to blocking with 5% skim milk in PBS, they were incubated overnight at 4°C with primary antibodies against deleted in azoospermia-like (DAZL; 1:1,800) [14 (link)] and NANOG (1:5,000; IHC-00205, Bethyl Laboratories, Inc., USA) in Can Get Signal immunostain (NKB-501; Toyobo Co., Ltd., Japan). After the samples were washed, they were incubated with Alexa 488- or Alexa 594-conjugated IgG antibodies at 25°C. The sections were enclosed in Gel/Mount (Biomeda Corp., USA) and observed using fluorescence microscopy (Axio Imager M2, Carl Zeiss, Germany). The number of germ cells in each genital ridge section was assessed by Image J (version 1.50i).

Lung tissues were fixed with 4% paraformaldehyde and sectioned into 5-μm-thick slices. Sections were blocked with 5% BSA and incubated with anti-mouse SP-A and anti-acrolein monoclonal antibodies as described above. Then, the sections were washed and incubated with secondary Alexa 488-conjugated anti-rabbit IgG or Alexa 568-conjugated anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA). The primary and secondary antibodies were diluted in immunoreaction enhancer solution (Can Get Signal immunostain, Toyobo Life Science). The cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Sections were analysed by confocal microscopy (Nikon A1, Nikon, Tokyo, Japan).

Kidney samples were collected in cryotubes and snap frozen in liquid nitrogen. Tissues were trimmed using cryostat blade (Leica, Cat. #14035838926) and then embedded in the OCT compound and frozen. Sections (15 μm) were fixed in 4 % paraformaldehyde at 4 °C for 15 min. After blocking with 10 % goat serum/PBS for 1 h, SLC12A1 and Tamm-Horsfall glycoproteins were detected with rabbit anti-SLC12A1 (2.5 μg/mL, Abcam, Cat. #ab191315) and sheep anti-Tamm-Horsfall glycoprotein (1:1000, Millipore, Cat. #AB733) at 37 °C for 1 h and subsequently at 4 °C for 16 h. Immunoreactivity was detected with TRITC-anti-rabbit IgG antibody (Jackson ImmunoResearch, Cat. #025-144-111) and FITC-anti-sheep IgG antibody (Jackson ImmunoResearch, Cat. #213-542-177). Both the 1st and 2nd antibodies were diluted with solution A of Can Get Signal immunostain (Toyobo, Cat. # NKB-501). Sections were counter-stained with a DAPI (Molecular Probe, Cat. #D1306) and examined under a confocal microscope (FV1000; Olympus Optical). Confocal images were analyzed by plot profile (Image J) [52 ] to semi-quantify the relative fluorescence intensity.