3 3 diaminobenzidine tetrahydrochloride substrate

NFs were harvested and fixed for 2 h in 10% buffered formalin at room temperature and prepared for paraffin embedding. To detect calcium deposition, sections were stained with 5% silver nitrate solution (ID Labs Biotechnology, Buffalo, NY), exposed to ultraviolet light for 30 min, and stained with nuclear fast red solution (ID Labs Biotechnology). To detect matrix proteoglycans, sections were stained with 0.1% Safranin O solution (Muto Pure Chemicals, Tokyo, Japan) and counter-stained with hematoxylin.
The primary antibodies used for immunohistochemistry included monoclonal mouse anti-human runt-related transcription factor 2 (RUNX2; H00000860-M06, Abnova, Taipei, Taiwan, 1: 100 dilution), monoclonal mouse anti-human osteocalcin (MAB1419, R&D, Minneapolis, MN, 1: 1000 dilution), monoclonal mouse anti-human dentin matrix protein-1 (DMP-1; sc-73633, Santa Cruz, Dallas, TX, 1: 100 dilution), and polyclonal rabbit anti-human type-II collagen antibody (ab34712, Abcam, Cambridge, MA, 1: 100 dilution). The secondary antibodies were horseradish peroxidase (HRP)-conjugated antibodies (Simple stain MAX PO, Nichirei, Tokyo, Japan). Antigens were visualized using a 3,3-diaminobenzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark) and counter-stained with hematoxylin.

The primary antibodies used for Western blotting or immunohistochemistry were monoclonal β-actin (AC-15, 1:1000; Santa Cruz), FGF23 (bs-5768R, 1:200; Bioss), αKlotho (NBP1-76511, 1:500; Novus Biologicals), NHERF1 (A-7, 1:200; Santa Cruz), NaPi-2a (ab182099, 1:500; Abcam), and NaPi-2a (ab151129, 1:500; Abcam), as described previously (Kang et al., 2017 (link)). Antigen retrieval for IHC was performed in Tris/EDTA (pH 9.0) for 2–3 min. IHC was performed with the following antibodies at 25°C for 2 hr: α-SMA (ab5694, 1:100; Abcam), NaPi-2b (S ab182099, 1:100; Abcam), TMPV6 (NBP2-32372, 1:100; Novus Biologicals), MINPP1 (LS-C368910, 1: 100; LSBio), or FGF23 (bs-5768R, 1:200; Bioss), Horseradish peroxidase-conjugated secondary antibodies were detected with 3,3,-diamino-benzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark). All sections were counterstained with hematoxylin (Themo Scientific).

Representative sections (n = 8) of pancreatic adenocarcinoma were obtained from Texas Cancer Research Biobank. 4 males and 4 females were studied (age 40–69). Sections were dewaxed in xylene and rehydrated through graded ethanol. After antigen retrieval with PT module (Thermo Scientific) for 17 minutes at 97°C, sections were incubated in 3% H₂O₂ in 50% methanol for 30 min at room temperature to quench endogenous peroxidase. To block nonspecific binding, sections were incubated in PBS containing 1% BSA and 0.1% Nonidet P-40 for 1 h at room temperature. Primary antibody reactions were incubated at 4°C overnight. Goat anti-LXRβ and anti-LXRα antibodies were developed as previously described [12] , [19] (link) and used at 1∶50 dilution in 1% BSA and 0.1% Nonidet P-40. Negative controls were incubated with PBS containing 1% BSA and 0.1% Nonidet P-40 without primary antibody. After washing, sections were incubated with goat-probe (Biocare Medical, GHP516) for 15 minutes, then washed in PBS and incubated with goat-on-rodent-HRP polymer (Biocare Medical, GHP516) for 15 minutes. After washing in PBS, sections were developed with 3,3′-diaminobenzidine tetrahydrochloride substrate (DAKO) and then counterstained with Mayer's hematoxylin. Sections were dehydrated through a graded ethanol series and xylene and finally mounted.