Can get signal immunostaining solution a

Musculus longissimus lumborum was paraffin-embedded and sectioned at a 5-μm thickness, as described previously [17 (link)]. Paraffin sections were deparaffinized with a lemosol solution (Fujifilm Wako Chemicals, Osaka, Japan). Collagen fiber staining was performed using a Picro-Sirius Red stain kit (ScyTek Laboratories, Logan, UT, USA).
For tissue immunostaining, deparaffinized sections were activated with an antigen-retrieval solution (Histo VT one solution; Nacalai Tesque, Kyoto, Japan) at 90 °C for 20 min. After permeabilization with 0.3% Triton-X in PBS (−) for 30 min and inactivation of endogenous peroxidase with 0.3% H₂O₂ solution for 30 min, the sections were treated overnight with an antibody diluted with Can Get Signal immunostaining solution A (Toyobo). The bound antibody was visualized using Histofine Max-Po (Nichirei Biosciences, Tokyo, Japan) and ImmPACT DAB (Vector Laboratories, Burlingame, CA, USA). Counterstaining was performed with Mayer’s hematoxylin solution (Fujifilm Wako Chemicals), and image analysis was performed using an all-in-one microscope system (BZ8000; Keyence, Osaka, Japan).

Cells were seeded in four-well glass chambers (Iwaki, Shizuoka, Japan) manually coated with collagen. Cells were fixed with 4% paraformaldehyde in 1× PBS for 15 min at RT and permeabilized with 0.1% Triton-X in 1× PBS for 20 min at room temperature. Primary antibodies diluted 1:100 in Can Get Signal Immunostaining Solution A (Toyobo, Osaka, Japan) were applied, and the samples were incubated overnight at 4 °C. Cells were incubated with the corresponding secondary antibodies conjugated to Alexa 488 (Molecular Probe, Eugene, OR, 1:500) for 1 h at room temperature and mounted using fluorescence mounting medium containing 4′,6-diamidino-2-phenylindole (Dako, Glostrup, Denmark). Images were captured under an Olympus DP72 microscope equipped with a digital camera using Olympus DP2-Twain software. The antibodies used were as follows: anti-STAT1 (#14994, CST, 1:100), -STAT2 (#72604, CST, 1:100), -IRF9 (#76684, CST, 1:100), and -8-OHdG (#MOG-20P; JaICA, Shizuoka, Japan).

Cells were seeded in 4-well glass chambers (IWAKI) with manual collagen coating. Cells were fixed with 4% PFA in 1 × PBS for 15 min at RT and permeabilized with 0.1% Triton-X in 1 × PBS for 20 min at room temperature. Primary antibodies diluted by 1:100 with Can Get Signal Immunostaining Solution A (Toyobo, Osaka, Japan) were applied to the samples and incubated overnight at 4 °C. Cells were incubated with the second antibodies conjugated with Alexa 555 or 488 (Molecular Probe, Eugene, OR, USA, 1:500) for 1 h at room temperature and mounted using fluorescence mounting medium with DAPI (Dako). Images were captured under an Olympus DP72 microscope digital camera system using the Olympus DP2-TWAIN software. The antibodies used were as follows: YBX1 (D299, CST, 1:100), α-tubulin (#3873, CST, 1:100), γ-tubulin (#T6557, Sigma-Aldrich, 1:100), RBBP6 (#NBP1–49535, Novus Biologicals, Littleton, CO, USA, 1:100), CTCF (#3418, CST, 1:400), ANKRD2 (#sc-138111, Santa Cruz Biotechnology, 1:100) and p53 (sc-6243, Santa Cruz Biotechnology, 1:100). Full-length blot images are available in Supplementary Fig. 11.