Anti collagen 1 antibody

Primary antibodies for Western blot analysis were as follows: anti-LOX1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STRA6 antibody (ABGENT, San Diego, CA), anti-CRBP1 antibody (Santa Cruz Biotechnology), anti-RARα antibody (Santa Cruz Biotechnology), anti-RARγ antibody (Santa Cruz Biotechnology), anti-RXRα antibody (Santa Cruz Biotechnology), anti-c-Jun N-terminal kinase (JNK) antibody (Santa Cruz Biotechnology), anti-pJNK antibody (Abcam, Cambridge, MA), anti-p38MAPK antibody (ABGENT), anti-p-p38MAPK antibody (ABGENT), anti-pSmad2 antibody (Santa Cruz Biotechnology), anti-Smad2 antibody (Santa Cruz Biotechnology), anti-TGFβ₁ (Santa Cruz Biotechnology), anti-caspase 3 antibody (Santa Cruz Biotechnology), anti-collagen 1 antibody (Santa Cruz Biotechnology), and anti-actin antibody (Millipore, Temecula, CA). Secondary antibodies for Western blot analysis as HRP-conjugate antibody were purchased from Millipore. The inhibitors of JNK, SP600125 and p38MAPK, SB203580 were purchased from Sigma-Aldrich (St. Louis, MO).

After deparaffinization and rehydration, kidney sections of saline-, L1-, or L5-injected mice, or L5-injected LOX1^−/− mice were placed in 0.01 M sodium citrate buffer (pH 6.0) and heated in a microwave oven for 2.5 min at 720 W. For Mason’s trichrome stain, sections were stained according to the protocol of the manufacturer (Sigma-Aldrich). For IHC, sections were washed in PBS and incubated with 1% BSA for 30 min to block nonspecific staining. Sections were drained and incubated for 3 h at room temperature in a humidity chamber with respective antibody for IHC, including anti-STRA6 antibody (ABGENT), anti-collagen 1 antibody (Santa Cruz Biotechnology Inc.), or anti-vitamin A antibody (MyBioSource, San Diego, CA) diluted with antibody diluent (Dako, Carpentaria, CA). After washing in PBS, endogenous peroxidase activity was blocked by incubation in 0.3% H₂O₂ in methanol for 20 min, followed by sequential 10 min incubations with biotinylated link antibody and peroxidase-labeled streptavidin (Dako). Staining was completed after incubation with 3,3’-diaminobenzidine substrate-chromogen solution (Dako), and then counterstained with hematoxylin. Images from similar regions of sections of kidney were captured by bright field microscopy at 400× microscopic magnification.

Proteins were extracted from the harvested hind limbs of 3-week-old rats. Isolated femurs and tibias were snap-frozen by adding liquid nitrogen on a mortar and homogenized using a pestle. Pulverized samples were incubated in the red blood cell lysis buffer (155 mM NH₄Cl, 140 mM NaHCO₃, and 0.1 mM EDTA, pH 7.3) for 10 min, and supernatants were removed by centrifuge. Pellets were lysed in ice-cold RIPA buffer containing protease inhibitor cocktails (Roche Applied Science). A controlled amount of proteins was separated using SDS-PAGE and transferred onto a PVDF membrane using an electroblot. After blocking with 5% milk TBS-T, the blots were probed using primary antibodies (anti-ZIP8 antibody, Pierce Biotechnology, Inc.; anti-Runx2 antibody, Sigma-Aldrich Co.; anti-β-actin antibody, Cell Signaling Technology, Inc.; and anti-Collagen-1 antibody, Santa Cruz Biotech.) and followed by a horseradish peroxidase-conjugated secondary antibody. Chemiluminescent reagents were used to detect immunoreactive proteins, and the membrane was exposed to X-ray films.