Paraffin-embedded lung and kidney tissue sections were deparaffinized, and then immersed in 0.2% Triton X-100 for 45 min as described previously (8 (link)). Following blocking with 10% donkey serum (ab7475, Abcam Inc, Cambridge, MA) in PBS for 1 h, slides were immunostained with rabbit anti-SP-A antibody, anti-TLR-4 (ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas). For cell IF analysis, the cells were fixed with 4% paraformaldehyde, and examined with anti-TLR-4(ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas) and anti-Megalin antibody (sc-16478, Santa Cruz Biotechnology, Dallas, Texas) to confirm the types of cultured cells and to determine the purity and quantity of proximal tubular epithelial cells. Slides were stained using Alexa 488 (ab150073, Abcam Inc, Cambridge, MA) and/or Alexa 594-conjugated secondary antibodies (A11058, Life Technologies, Eugene, OR) at room temperature for 1 h for fluorescence visualization of primary antibodies.
Chen X., Guo J., Mahmoud S., Vanga G., Liu T., Xu W., Xiong Y., Xiong W., Abdel-Razek O, & Wang G. (2023). Regulatory roles of SP-A and exosomes in pneumonia-induced acute lung and kidney injuries. Frontiers in Immunology, 14, 1188023.
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