Anti laminin

Cleared samples were incubated in PBST for 2 h and then blocked with 2% bovine serum albumin (BSA; Sigma‐Aldrich Inc., St. Louis, MO, USA) in PBST for 6 h. The samples were incubated for 24–72 h with the following primary antibodies: anti-OPG (Biorbyt, Cambridge, United Kingdom), anti-RANKL (Biorbyt, Cambridge, United Kingdom), anti-RUNX2 (Santa Cruz Biotechnology Inc., TX, USA), anti-COL-1 (Abcam Inc., Cambridge, United Kingdom), anti-COL-4 (Abcam Inc., Cambridge, United Kingdom), anti-laminin (Santa Cruz Biotechnology Inc., TX, USA) and anti-CD31 (Santa Cruz Biotechnology Inc., TX, USA). The samples were then washed three times in PBST for 24–48 h, followed by incubation with the secondary antibodies and lectin dye (Lycopersicon esculentum (Tomato) Lectin (LEL, TL), DyLight® 594; Vector Laboratories, CA, USA) in PBST for 24–72 h. Secondary antibodies were purchased as conjugates with Alexa Fluor 488/647 (Life Technologies, Darmstadt, Germany) and transferred to nRIMS solution in 50 mL tubes. Immunolabeled samples were washed three times with PBST for 24–72 h and stored in 5 mL nRIMS for 6–24 h. Each sample was incubated in nRIMS in a confocal dish and covered with a 24 mm diameter coverslip. Whole bone samples in nRIMS were sandwiched between two 24 × 60 mm coverslips and small 1-mm-thick magnets.

After rinsed with PBS and fixed with 4% paraformaldehyde for 10 min, cells were rinsed with PBS and permeabilized with 0.05% triton X-100 for 10 min. After incubated with 5% horse serum for 30 min and then with primary antibody over night at 4°C, the cells were incubated with secondary antibodies for 1 h at room temperature. The primary antibodies included anti-fibronectin (1 : 100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-laminin (1 : 100, Santa Cruz Biotechnology). The secondary antibody is Alexa-594-conjugated secondary antibodies (1 : 500; Life Technologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies).