3000b epifluorescence microscopy

After fixation with 4% paraformaldehyde, cell samples were treated with PBS containing 5% donkey serum and 0.2% Triton-X100 for 20–30 min at room temperature. Samples were incubated in the primary antibody at 4 °C overnight, followed by corresponding secondary antibody incubation at room temperature for 2 hr. Secondary antibodies included AMCA, Alexa Fluor 488, Cy3, and Alexa Fluor 647 conjugated donkey anti-mouse, goat, rabbit, or chicken antibodies (Jackson Immunoresearch). 4,6-Diamidino-2-Phenylindole (DAPI; Invitrogen) was used to label all nuclei. Samples were observed and imaged by Leica 3000B epifluorescence microscopy and/or Leica SPE confocal microscopy.
For western blotting, purified TSP1 protein, ACM collected from wild type Swiss Webster mice, TSP1-ID-ACM, mock-ID-ACM, TSP1-KO-ACM, and DMEM/F12 were loaded to blotting gels. Antibodies used in western blotting included goat anti-TSP1 antibodies, donkey anti-goat HRP-conjugated antibodies, and HRP standard protein (all from Bio-Rad). SuperSignal West Femto Stable Peroxide Solution and SuperSignal West Femto Luminol/Enhancer Solution (ECL, all from Thermo scientific) were applied to blotting membrane for protein detection. Images were captured using a ChemiDoc-It 2 imaging system (UVP).