Alexa fluor 488 conjugated anti rat igg antibody

After treatment, infection and fixation, coverslips containing attached cells were washed with PBS, incubated for 20 minutes with PBS containing 2% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) and processed for an inside/outside immunofluorescence invasion assay as described previously [27 (link)]. Briefly, cells were fixed and extracellular parasites were immune-stained using a 1:500 dilution of rabbit anti-T. cruzi polyclonal antibodies in PBS containing 2% (w/v) BSA (PBS/BSA) followed by labeling with Alexa Fluor-546 conjugated anti-rabbit IgG antibody (Invitrogen).
For evaluation of parasitophorous vacuole escape, after extracellular parasite staining, cells were permeabilized using a solution containing 2% (w/v) BSA and 0.5% (v/v) saponin (Sigma-Aldrich) in PBS (PBS/BSA/saponin) for 20 minutes. Host cell lysosomes were then immunostained using a 1:50 dilution of rat anti-mouse LAMP-1 hybridoma supernatant (1D4B; Developmental Studies Hybridoma Bank, USA) in PBS/BSA/saponin for 45 minutes followed by labeling with Alexa Fluor-488 conjugated anti-rat IgG antibody (Invitrogen), as described previously [30 (link)]. Subsequently, DNA from both host cells and parasites were stained for 1 min with 10 μM of DAPI (Sigma-Aldrich), mounted on glass slide and examined on an Olympus BX51, Zeiss, Apotome or Nikon Eclipse Ti.

Three-day-old seedlings of C24 and der1-3 mutant cultivated on half-strength MS medium were prepared for whole-mount immunofluorescence labelling of roots according to a standard protocol (Beck et al., 2010 (link); Šamajová et al., 2014 (link)) with some modifications. Samples were fixed at room temperature for 1 h or at 4 °C overnight. Washing of samples after aldehyde reduction was done five times for 10 min and washing after cell wall digestion was done three times for 10 min. For blocking we used 5 % w/v bovine serum albumin (BSA) in phosphate-buffered saline (PBS). As the primary anti-α tubulin rat monoclonal antibody we used clone YOL1/34 (Bio-Rad) diluted in 3 % w/v BSA, and thorough washing after incubating with the primary antibody was done 12 times for 10 min with 3 % w/v BSA in PBS. Subsequently, a secondary Alexa Fluor 488-conjugated anti-rat IgG antibody (Molecular Probes), appropriately diluted in 3 % w/v BSA, was applied and incubation was done at 37 °C for 3 h followed by incubation at 4 °C overnight. Nuclear DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich). Samples were examined with a confocal laser scanning microscope.