September 20, 2018
The Journal Of Biological Chemistry
Product: Alpha Toxin from C. septicum, Liquid
Indirect immunofluorescence assays:
HFF cells seeded on glass cover slips were infected with freshly isolated tachyzoites for 24 h, fixed with 4 % paraformaldehyde for 10 min, and then neutralized with 0.1 M glycine in PBS for 5 min. Samples were permeabilized by 0.1 % triton X -100 in PBS for 20 min, blocked with 2 % bovine serum albumin in PBS/0.1 % triton X -100 for 20 min. Subsequently, they were stained with primary antibodies (mouse anti -HA, 1:10.000; rabbit anti -TgGap45, 1:10.000) for 1 h and then with respective secondary antibodies (Alexa488 and Alexa594, 1:3.000) for 45 min in the dark. Samples were mounted into a mix of fluoromount G and DAPI, dried at 4 C overnight and examined by fluorescence microscopy (ApoTome, Zeiss). To resolve the C -terminal topology of TgFNT1 – 3, extracellular parasites were stained with a mouse -HA antibody (1:3000), suspended either in 3% BSA/PBS solution for pre -permeabilization staining, or in 2% BSA/0.2% triton X -100/PBS for post -permeabilization, as shown in Fig. S1. The inner membrane complex was separated from the plasma membrane by treating extracellular parasites with -toxin from Clostridium septicum (20 nM, 2 h) (List Biological Laboratories) followed by fixation on BSA -coated (0.01%) coverslips. In both cases, the standard secondary antibody staining procedure was performed afterwards, as described above.