Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

Double immunofluorescent microscopy (DIF) of S. suis infected PTEC and PBEC was performed as previously described 42 with some modifications. All incubation steps were performed at RT. All Infected cells were washed three times with PBS+ and fixed with 3.7% formaldehyde in PBS for 20 min. Formaldehyde-fixed preparations were washed and 0.1 M glycine in PBS was added for 5 min, after then samples were washed 3 times with PBS and followed by incubation in blocking buffer containing 1% bovine serum albumin (BSA) in PBS to block nonspecific binding sites. All antibodies were diluted in 1% BSA in PBS. Blocking buffer was removed and preparations were incubated with rabbit anti-S. suis antiserum diluted 1:200 for 1h. After washed with PBS, samples were incubated for 45 min with green fluorescent anti-rabbit IgG secondary antibody Alexa Fluor ® 488 anti-rabbit IgG (H+ L) antibody 1:1000 diluted (Life Technologies) to stain extracellular streptococci. Following, the preparations were washed with PBS and permeabilized with 0.2% Triton X-100 for 20 min. Then preparations were again incubated with the rabbit anti-S. suis antiserum 1:200 diluted for 1h, washed with PBS, then incubated with red fluorescent anti-rabbit IgG secondary antibody (Alexa Fluor ® 568 anti-rabbit IgG (H+ L) antibody Scientific RepoRts | 6:26748 | DOI: 10.1038/srep26748 1:1000 diluted (Life Technologies)) for 45 min to stain intra-and extracellular bacteria. After PBS washing steps, the preparations were incubated with DAPI (4′ ,6-diamidino-2-phenylindole) and after final washing, the membrane of transwell filters were cut down and embedded in Mowiol and stored at 4 °C for further analysis.