Lsm 7 duo

Thaw-mounted 20-μm coronal sections were cut on a cryostat at −15°C and mounted in gelatin-coated slides. For double-labeling of Iba-1 with CD16/CD32 (CD16/32), sections were blocked with PBS containing 1% normal goat serum and chicken serum (Vector Laboratories, Burlingame, CA, USA) for 1 h. The slides were washed three times in PBS and then incubated overnight at 4°C with either rabbit-derived anti-Iba-1 (1:300; Wako) or rat-derived anti-CD16/32 (1:500; BD Biosciences) diluted in PBS containing 1% normal goat or chicken serum and 0.25% Triton X-100. Sections were incubated with goat anti-rabbit secondary antibody conjugated to fluorescein (1:200, for Iba1; Vector) and chicken anti-rat secondary antibody conjugated to Alexa Fluor® 594 (1: 200, for CD16/32; Invitrogen) for 1 h at 22 ± 1°C in the dark. This addition was preceded by three 10-min rinses in PBS. As a control, another set of sections was incubated with only the Iba-1 antibody and then visualized using both filters. No signal was detected when Iba-1 alone with fluorescein filter was used (photomicrograph not shown). The same was true with CD16/32 when Alexa Fluor® 594 filter was used. Fluorescence images were acquired using a confocal laser scanning microscope (Zeiss LSM 7 DUO) and processed using its associated software package (ZEN 2010).

The 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence analysis was carried out to measure cytosolic pH. According to the previously described studies (Sundaresan et al., 2014 (link); Ying et al., 2016 (link)), Arabidopsis flowers were removed from the plant body and soaked into 10 μM BCECF-AM (B1150, Thermo Scientific) solution for 20 min under darkness. Then, phosphate-buffered saline (PBS, pH 7.4) was used to remove the excess BCECF-AM. Images were taken using the confocal laser scanning microscope (LSM 7 DUO, ZEISS, Germany). BCECF fluorescence and chlorophyll autofluorescence were detected under 488 nm and 633 nm light, respectively.