Primary anti laminin antibody

Recombinant human GH was purchased from Dong-A Pharmaceutical Co., Ltd (Dalseong-Gun, Daegu, South Korea). Laminin 111 protein, primary anti-laminin antibody and secondary goat anti-rabbit-FITC conjugated antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies: anti-CD4 allophycocyanin (APC), anti-CD8 peridinin chlorophyll protein (PerCP), anti-CD49f phycoerythrin (PE) and rat IgG2a PE were purchased from eBioscience (San Diego, CA, USA). Fetal bovine serum (FBS), RPMI-1640 medium, l-glutamine, gentamicin, Trypsin–EDTA, phosphate buffered saline (PBS) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich.

VDR mRNA spatial distribution in muscle cross sections was assessed with RNAscope in situ hybridization to visualize VDR RNA following the manufacturer’s guidelines (Advanced Cell Diagnostics [ACD]). Briefly, sections were cut at 7-μm thickness and stored at –80°C to preserve mRNA integrity before in situ hybridization. Sections were fixed in 4% PFA for 15 minutes, ethanol dehydrated, and incubated in H₂O₂ for 10 minutes to quench endogenous peroxidases. Antigen retrieval was performed using a protease (ACD, 322336). Target mRNA was hybridized with a human VDR probe (ACD, 530961), amplified, and detected using the Opal 570 fluorescent reagent (ACD, 323272). Samples were incubated overnight in anti-laminin primary antibody (Sigma-Aldrich, L9393). Secondary antibody (Thermo Fisher Scientific, A32790) incubation occurred the following day and sections were subsequently DAPI stained.
VDR RNA in situ hybridization images were acquired using a Zeiss LSM 880 upright confocal microscope equipped with an Airyscan detection unit and an argon laser. Imaging was conducted with a 20× (Plan-Apochromat, NA 1.0, water) or a 63× (Plan-Apochromat, NA 1.4, oil) objective lens. DAPI was excited at 405 nm, GFP at 488 nm, and Opal 570 at 561 nm.