Rabbit anti fibronectin antibody

Rabbit anti-collagen I antibody was purchased from Rockland antibodies (#600-401-103-0.5) and used at 1:2,000. Rabbit anti-collagen IV (ab6586) and collagen VI (ab6588) antibodies were from Abcam and used at 1:3,000 dilution. The antibodies against the alpha chains of mouse collagen VI were a kind gift of Raimund Wagener39 (link) and used at 1:500. Rabbit anti-fibronectin antibody was from Sigma (F3648) and used at 1:1,000 dilution. Rabbit anti-MMP2 antibody was from Abcam (ab37150) and used at 1:2,000 dilution. Mouse anti-MMP14 antibody was from Millipore (MAB3328) and used at 1:3,000 dilution. Rabbit β-arrestin 1 antibody was from Thermo Fischer Scientific (PA5-19582) and used at a 1:1,000 dilution. Mouse anti-actin antibody was from Millipore (MAB1501) and used at a 1:3,000 dilution. Anti-phospho-Tyr (clone 4G10) was from Upstate and used at 1:500. Mouse anti-V5 antibody were purchased from Invitrogen and used at a 1:2,000 dilution and rabbit anti-V5 antibody from Covance and used at a 1:3,000 dilution. Anti-mouse or human CMG2/ANTXR2 are monoclonal antibodies developed by rat genetic immunization and purified from hybridomas supernatant (Genovac) both used at 1:2 dilution. Horseradish peroxidase-conjugated secondary antibodies were from Pierce and used at 1:2,000 dilution. Protein G beads were purchased from GE Healthcare.

Immunofluorescent staining was performed as described previously [21] (link). The expression of VDR in cultured renal cells was detected using rat anti-VDR antibody (Abcam, Cambridge, UK) followed by Alexa Fluor 488 goat anti-rat IgG (Invitrogen, Carlsbad, CA). Similarly, PPARδ and COUP-TFII were detected using rabbit anti-PPARδ (Affinity Bioreagents, Golden, CO) and anti-COUP-TFII antibody (Abcam) followed by Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen). Renal expression of NOR1 was detected using mouse anti-NOR1 antibody (Abcam) followed by Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). To determine whether NOR1 was localized in mesangial cells, podocytes, proximal tubular epithelial cells, or collecting duct cells, the sections were counter-stained with rabbit anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-WT-1 antibody (Abcam, Cambridge, UK), rabbit anti-aquaporin 1 (AQP1) antibody (Millipore, Temecula, CA), or rabbit anti-AQP2 antibody (Abcam) respectively, followed by Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen). Fluorescence images were obtained using a fluorescence microscope (BX51; Olympus, Tokyo, Japan).

Recombinant human UCHL1 protein and rabbit monoclonal antibody (R&D Systems, USA), recombinant human leptin protein and rabbit anti-leptin antibody (Abcam, United Kingdom), fibronectin from human plasma and rabbit anti-fibronectin antibody (Sigma-Aldrich, Germany) were used.
The reagents described in the previous study^{16 (link)} were utilized in the current study. „The cysteamine hydrochloride, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccimide (NHS) were from Sigma-Aldrich, Germany, absolute ethanol, acetic acid, hydrochloric acid, sodium hydroxide, sodium chloride, sodium carbonate, sodium acetate were from POCh, (Poland) HBS-ES buffer pH = 7.4 (0.01 M HEPES, 0.15 M sodium chloride, 0.005% Tween 20, 3 mM EDTA), Phosphate Buffered Saline (PBS) pH = 7.4, carbonate buffer pH = 8.5 from BIOMED (Poland) were used as received. Aqueous solutions were prepared with Milli-Q water (Simplicity® Millipore). Argon N 5.0 with a content Ar ≥ 99,999% was used (AIR LIQUIDE Polska Sp.z o.o., Poland).”

All reagents were purchased from Sigma (Poole, UK) unless otherwise specified. Frozen tissue sections were left for 30 min to equilibrate to room temperature and then hydrated in PBS 3 × 5 min before being immersed in 0.1% Triton X-100 for 20 min to permeabilise the sections. Following a further PBS 3 × 5 min wash, eye sections were isolated with a hydrophobic PAP pen (Vector Laboratories, Peterborough, UK). Non-specific antibody binding sites were blocked using 0.5% BSA, 0.3% Tween-20 and 15% Normal Goat Serum in PBS. Sections were then placed in primary antibody (1/200 rabbit anti-laminin antibody, L9393, Sigma; 1/200 rabbit anti-fibronectin antibody, F3648, Sigma; 1/100 goat anti-Brn3a antibody, SC-31984, Santa Cruz Biotechnology, Dallas, US) and left overnight at 4 °C before being washed 3 × 5 min in PBS and incubated for 1 h at room temperature with secondary antibody (1/400 goat anti-rabbit Alexa Fluor 594, A-11058, or 1/400 donkey anti-goat Alexa Fluor 488, A-11012, both from Thermo Fischer Scientific). Before mounting with Vectashield containing DAPI (Vector Laboratories), sections were washed 3 × 5 min in PBS. Control tissue sections, incubated without primary antibodies but with secondary antibodies, were all negatively stained (not shown).