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).
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).
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