Wga conjugated to alexa488

For analysis of MHC-I downregulation, cells were seeded, transfected and fixed as described above (paragraph 2.5). Afterwards, cells were incubated with primary rabbit anti-FLAG antibody (Sigma-Aldrich) at 1:300 and mouse anti human HLA-A, B, C class I antibody (1:100) in HEK-293 cells (Santa Cruz) or mouse anti-pig SLA class I antibody (1:100) for PK-15 cells (R&D systems) for 1 hour at RT. Following three washes with PBS/CaCl₂, cells were incubated with secondary goat anti-rabbit Alexa Fluor 647 antibody and goat anti-mouse Alexa Fluor 555 antibody at 1:500 in PBS for 1 hour at RT. For the last 10 minutes of the incubation, WGA conjugated to Alexa488 (Invitrogen) was added to the cells as a membrane marker (5μg/mL). Following two wash steps, cells were incubated with PBS containing Hoechst 33342 stain (Invitrogen) (1μg/mL). Before imaging on a fluorescence microscope (LSM700) cells were mounted with 8μL of Fluorescence mounting medium (Dako). Equivalent numbers of transfected and non-transfected cells were blindly selected and we measured the intensity of 555 channel (MHC-I) using FIJI software.

The observation of necrotic death area hyphae and H₂O₂ detection assay were performed as previously described [26 (link)]. Leaf segments were fixed and decolorized in a mixture of acetic acid/ethanol (1:1) for 3 d. Autofluorescence of mesophyll cells was observed to determine necrotic death area using epifluorescence microscopy (excitation filter, 485 nm; dichromic mirror, 510 nm; barrier filter, 520 nm). H₂O₂ accumulation was detected by staining with DAB (Amresco, Solon, OH, USA). Hyphae were stained with WGA conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA) and observed under blue-light excitation (excitation wavelength 450–480 nm, emission wavelength 515 nm). Only the site where an appressorium had formed over a stoma was considered to be a successful penetration. The H₂O₂ accumulation, necrotic areas, hyphal length, and hyphal areas were observed with a BX-53 microscope (Olympus) and calculated using DP-BSW software.