Blocking one solution

Tissue samples were fixed in 4.0% PFA/PBS at 4°C overnight. Samples were permeabilized using 0.5% Triton X-100/PBS at room temperature for 30 min. Endogenous peroxidase was inactivated by 3.0% hydrogen peroxide/PBS for 10 min. Then, the sample was treated with Blocking-One solution (Nacalai Tesque) at room temperature for 1 h. Primary antibody (anti-Cubn) was used at a 1:500 dilution with Blocking-One solution. Samples were incubated with primary antibody at 4°C overnight. Secondary antibody was used at a 1:500 dilution in 0.1% Tween-20/PBS with 4′,6-diamidino-2-phenylindole (DAPI). Samples were treated with the secondary antibody solution at 4°C overnight. Microscopic observation was performed using a Leica TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany).

Serial sections of 3-μm thick paraffin-embedded testis were prepared and mounted on poly-L-lysine slides. The sections were deparaffinized in xylene and then rehydrated in graded alcohols.
The tissue sections were heated in a microwave oven in pre-heated 10 mM sodium citrate buffer pH 6.0 for 5 min for antigen retrieval and permeabilized with 0.1% Triton-X 100 in PBS for 30 min. The slides were washed three times with PBS before incubation in Blocking One Solution (Nacalai Tesque, Kyoto, Japan) for 10 min to block non-specific binding. The slides were rewashed with PBS three times. The tissue sections were incubated overnight at 4°C with mouse monoclonal phosphohistone 3 (pHH3) antibody conjugated with Alexa Flour 594 (Santa Cruz Biotechnology, Texas, United States) at a dilution of 1:100.
After antibody binding, the slides were washed three times with PBS. DNA was counterstained with 10 μg/mL Hoechst 33342 and observed using Nikon Eclipse Ni fluorescent microscope under 400 × magnification. About 10–15 random fields were captured using Nikon Y-T TV. The intensity of the staining of 20 random seminiferous tubules was measured using Image J software v1.52a. The steps were repeated for rectum adenocarcinoma tissue (Kim et al., 2018 (link)) as a positive control for the staining.

CD29⁺ cells in the glass chamber or muscle-sectioned tissue were fixed in 4% paraformaldehyde for 10 min. After washing with PBS, cells were permeabilized with 0.2% Triton-X/PBS for 5 min and blocked with Blocking One solution (Nacalai Tesque) for 1 h. Subsequently, CD29+ cells were stained with monoclonal anti-desmin (mouse IgG, 1:100 dilution, #D1033; Sigma-Aldrich). Muscle sectioned tissue was stained with monoclonal anti-Pax7 (mouse IgG1κ, 1:50 dilution, #sc-81648; Santa Cruz, CA, USA) and APC-conjugated anti-CD29 (Ha2/5) (BD Biosciences). Primary antibodies were incubated with 2% BSA/PBS at 4 °C overnight. Cells or sectioned tissue were washed with PBS twice and stained with Alexa Fluor 594-conjugated anti-mouse IgG1 second antibody (1:1000, Life Technologies), Alexa Fluor 488-conjugated anti-mouse IgG1 second antibody (1:1000, Life Technologies), and Hoechst 33258 (1:1000, Dojindo, Kumamoto, Japan) for nuclear staining. After washing with PBS twice, cells were mounted using PermaFluor™ Adqueous Mounting Medium (Thermo Fisher Scientific) and visualized using a BZX-710 microscope (Keyence, Osaka, Japan).

Western blotting was performed as described previously with some modifications^{18 (link)}. Briefly, cells were lysed with RIPA buffer (Wako) containing a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein (10 μg) were electrophoresed using 10% sodium dodecyl sulphate–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, washed with Tris-buffered saline containing 0.05% Triton X-100, and incubated with BlockingOne solution (Nacalai Tesque, Kyoto, Japan) for 60 min. Anti-p62 (1:1,000; MBL) and anti-ubiquitin (1:1,000; Santa Cruz Biotechnology) antibodies were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Promega, Madison, WI, USA) were used as the secondary antibodies. As a control, β-actin was detected with anti-β-actin pAb-HRP-DirecT (1:2000; MBL). Blots were visualized using Chemi-Lumi One L (Nacalai Tesque). Stained membranes were scanned with ImageQuant LAS 4000 (GE Healthcare, Menlo Park, California, USA). Quantification was performed using ImageJ software (https://imagej.nih.gov/ij/).

Protein aliquots of 25 μg each were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were washed three times and then incubated with Blocking One solution (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against anti-RET (D3D8R) (#14698), anti-phospho-RET (Tyr905) (#3221), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#4060), anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9664), and anti-β-actin (13E5) (#4970) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA). Additional antibodies were also used including anti-human/mouse/rat extracellular signal-regulated kinase (ERK) 1/ERK2 (0.2 μg/mL) (AF1576) and anti-phospho-ERK1/ERK2 (T202/Y204) (0.1 μg/mL) (AF1018) from R&D Systems. The membranes were washed three times and then incubated for 1 hour at room temperature with species-specific horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Each experiment was performed independently at least three times.

Tissue lysates and a plasma membrane fraction were prepared from the skeletal muscle as described previously [11 (link)]. After the determination of protein concentration, aliquots of tissue lysates containing 30 μg of protein and a plasma membrane fraction containing 5 μg of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked using Blocking One^® solution (Nacalai Tesque, Kyoto, Japan) for 2 h at room temperature, incubated with primary antibodies overnight at 4 °C, and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The proteins were visualized using an ImmunoStar^® LD and Light-Capture II (ATTO Corp., Tokyo, Japan). The density of the specific band was determined using ImageJ image analysis software (National Institutes of Health, Bethesda, MD, USA).