Anti col4

From each sample, 16–20 mg of protein was extracted and utilized for Western blot. The antibodies utilized in this research included anti-FN (1:1000; Abcam; HK), anti-Col-4 (1:1000; Abcam; HK), and anti-TGFβ1 (1:1000; Abcam; HK). GAPDH (1:2000; Abcam; HK) was utilized as the loading control.

The impact of AS-IV on kidney fibrosis in DN rats was examined by Western blot assays. Briefly, renal tissues from individual rats were homogenized by RIPA buffer containing phosphatase and protease inhibitors and centrifuged. After quantifying the protein concentrations using BCA method, the tissue lysates were separated by SDS-PAGE on 8-10% gels and electrically transferred onto PVDF membrane. The membrane was sealed by 5% skimmed milk in TBST buffer and incubated overnight at 4°C with anti-COL4 (Abcam, UK, 1 : 1000), anti-FN (Proteintech, 1 : 1000), and GAPDH (Servicebio, China, 1 : 3000). The bound antibodies were detected with HRP-conjugated secondary antibodies and visualized using the enhanced chemiluminescent reagent. The data were quantitated by densitometry using ImageJ software.

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.