Sp8 laser scanning confocal microscope system

IF was performed on FFPE sections after heat-induced antigen retrieval. Briefly, sections were blocked for 1 h, incubated overnight at 4°C with a rabbit primary antibody against human CACNG4 (Immunoway, YM3404), and subsequently incubated with a secondary Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Abcam, ab175696) for 1 h. Slides were mounted with mounting medium containing DAPI and imaged by a Leica SP8 laser scanning confocal microscope system. For WB, samples were pooled and extracted using the FFPE Total Protein Extraction Kit (Sangon Biotech, C500058) and then incubated with primary antibody against CACNG4 or β-actin (Cell Signaling, #3700). The proteins were visualized using HRP-conjugated secondary antibody (Cell Signaling, #7040 or #7076) and chemiluminescent HRP substrate (Bio-Rad).

Brain samples from the experimental animals were fixed in 10% formalin in PBS, dehydrated in graded ethanol, and embedded in paraffin before obtaining 4-µm sections for further experiments, including hematoxylin and eosin staining, immunofluorescence assays, and immunohistochemical assays. The EV-A71 antigen was detected using a primary mouse anti-EV-A71 monoclonal antibody (Chemicon, USA) and a secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Sigma, Germany) in immunohistochemical analyses or an Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody (Life Technologies, USA) in immunofluorescence assays. Astrocytes were detected using a rabbit anti-GFAP antibody (Abcam Ltd., UK) as the primary antibody and an Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (Life Technologies, USA) as the secondary antibody. The staining procedure was performed according to a standard protocol [15 (link), 18 (link)]. The histopathological and immunohistochemical analyses were performed using a light microscope (Nikon DS-Ril/Eclipse), and the immunofluorescence assay was performed with a Leica SP8 laser scanning confocal microscope system.

Minimal medium (MM) [22 (link)] was used for penetration assays. The Vda^Sm cultures were incubated on a cellophane membrane (DINGGUO, Beijing, China), which was overlaid onto MM. To determine if any Vda^Sm penetrated the cellophane, the hyphae were observed in the medium after removing the membranes. The experiments for each colony were repeated independently at least three times. For hyphopodium detection, the mycelium was grown on cellophane for 2 days and observed as previously described [22 (link)]. The protocol of plasma membrane staining using FM4-64 (ThermoFisher, Shanghai, China), ER-Tracker (ThermoFisher, Shanghai, China) staining and protein localization assays have been described [22 (link),31 (link)].
To observe the infection process of Vda^Sm, eggplant roots (Hang No. 1) were inoculated for 8 days and sectioned. Fluorescent photographs were captured using a Leica SP8 confocal laser scanning microscope system [21 (link),22 (link)].