Mouse anti goat

An indirect ELISA was set up to detect FIPV antibodies in the cat sera. A total of 116 samples were tested, including all 19 serum samples positive for N-ELISA and additional 97 randomly selected serum samples to evaluate the seroprevalence of FIPV in these pet cats. The native FIPV antigens (catalog # MBS568874, MyBioSource) were used to coat the plate at 200 ng/well in bicarbonate coating buffer at 4°C overnight. The plate was blocked with 5% nonfat milk in PBST for 1 h at 37°C. Heat-inactivated cat serum samples were diluted at 1:50 in PBST with 1% nonfat milk and added into both FIPV-coated and un-coated wells. Goat anti-FIPV polyclonal antibody (catalog # MBS560925, MyBioSource) was included as a positive control. The HRP-conjugated goat anti-cat (Cat # RL602-1302; Rockland Immunochemical) and mouse anti-goat (cat # sc-2354, Santa Cruz) secondary antibodies were used at 1:1000 dilution in PBST with 1% nonfat milk. After 10 min – 15 min of substrate incubation at 37°C, the stop solution (3 N HCl) was added. Normalized OD₄₅₀ was obtained and analyzed as described above.

For Western blot analysis, rat urine, cell culture supernatants, and cell lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were routinely stained by Ponceau S (Sigma Aldrich Inc.). The primary antibodies were rabbit anti-rat MMP-9 (1:3000), rabbit anti-megalin (1:1000, Abcam), mouse anti-TGF-β1 (1:250, Santa Cruz), and mouse anti β-actin or α–tubulin (1:5000; Sigma Aldrich Inc.). HRP-conjugated secondary antibodies were goat anti rabbit (1:5000), mouse anti-goat (1:5000) and goat anti-mouse (1:5000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Detection was accomplished using enhanced chemiluminescence Western blotting (ECL, GE Healthcare, Piscataway, NJ, USA). For urine and cell culture supernatants, Ponceau red staining was used for loading control. Either β-actin or α-tubulin was used as an internal control for cell lysates. Relative band intensity was measured densitometrically using ImageJ software.