We produced rat monoclonal rCOL4A6, as described previously49 (link),50 (link). Briefly, the antigen emulsion was injected to Col4a5 deficient males. The treated rats were sacrificed 21 days after the injection, and the lymphocytes were fused with SP2/0-Ag14 myeloma cells. After the cell fusion, culture supernatants were screened to confirm positive clones by solid-phase enzyme-linked immunosorbent assay (ELISA).
The following primary antibodies were used: rat monoclonal type IV collagen α1(IV) (H11), α2 (IV) (H22), α3 (IV) (H31), α4 (IV) (H43), α5(IV) (H52) (In our institute)3 (link); Tubulin (T9026), α-SMA (1A4) (Sigma, St. Louis, MO); phospho-Smad3 (ab52903), Fibronectin (ab6328), LDL receptor (ab52818), MMP12 (ab52897) (Abcam, Cambridge, UK); Smad3 (#9523, Cell Signaling Technology, Beverly, MA); Nephrin (GP-N2, Progen, Germany); Nestin (66259-1-Ig, proteintech, Chicago, IL); Laminin β2 (MAB2066), OPN (AF808) (R&D systems Minneapolis, MN); CTGF (sc-373936), MMP3/10 (sc-374029) (Santa Cruz Biotechnology, Santa Cruz, CA); and MBP (New England Biolab). Primary antibodies were detected using species-specific secondary antibodies conjugated to either Alexa Fluor 488 or 555 (Molecular probes).
The following primary antibodies were used: rat monoclonal type IV collagen α1(IV) (H11), α2 (IV) (H22), α3 (IV) (H31), α4 (IV) (H43), α5(IV) (H52) (In our institute)3 (link); Tubulin (T9026), α-SMA (1A4) (Sigma, St. Louis, MO); phospho-Smad3 (ab52903), Fibronectin (ab6328), LDL receptor (ab52818), MMP12 (ab52897) (Abcam, Cambridge, UK); Smad3 (#9523, Cell Signaling Technology, Beverly, MA); Nephrin (GP-N2, Progen, Germany); Nestin (66259-1-Ig, proteintech, Chicago, IL); Laminin β2 (MAB2066), OPN (AF808) (R&D systems Minneapolis, MN); CTGF (sc-373936), MMP3/10 (sc-374029) (Santa Cruz Biotechnology, Santa Cruz, CA); and MBP (New England Biolab). Primary antibodies were detected using species-specific secondary antibodies conjugated to either Alexa Fluor 488 or 555 (Molecular probes).
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