Nestin 66259 1 ig

We produced rat monoclonal rCOL4A6, as described previously^{49 (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).

Similarly, three different cell types (CSF-cNs, SVZ-NSCs, and NE-4C) were washed with cold PBS for two times and centrifuged at 1,000 rpm for 5 min to obtain cell pellet. Then these cells were lysed in RIPA lysis buffer (RIPA, Beyotime, Shanghai, China). The protein concentration was measured with BCA assays (Thermo Fisher Scientific, Waltham, MA, United States). The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. We used 5% milk to block the membrane at room temperature for 90 min. PVDF membrane and the primary antibody were incubated overnight at 4°C. After washing with Tris-buffer saline tween (TBST) with three times, then the PVDF membrane was incubated with horseradish peroxidase (HRP) conjugated anti-mouse IgG and anti-rabbit IgG (1: 5000, Proteintech, Wuhan, China) for 1 h. Subsequently, we visualized these immunolabeled bands with an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, United States).
The primary antibodies included Nestin (66259-1-Ig,1:500, Proteintech, Wuhan, China), Pkd2l1 (13117-2-AP, 1: 500, Proteintech, Wuhan, China), Sox2 (66411-1-Ig,1: 1000, Proteintech, Wuhan, China), β-Actin (20536-1-AP, 1-10000, Proteintech, Wuhan, China).