Dmirb fluorescence microscope

The pollen sampled from the W.T. and mutant plants’ rapeseed buds immediately before flowering were stained with 1% (w/v) acetocarmine solution to analyze the pollen viability. Three biological replicates were used for this study. The stained pollen grains were visualized, and the images were recorded using a Leica DMIRB fluorescence microscope.

EPC cells were inoculated on a microscopic coverslip in 6-well plates grown to 90% confluence. Then, 1.25 μg of each recombinant plasmid were transfected into EPC cells using the lipofectamine 3000 (Invitrogen, MA, USA) reagent (according to the manufacturer’s instruction). At 24 h post-transfection (hpt), cells were fixed with 4% paraformaldehyde (PFA) for 30 min, permeabilized with 0.2% Triton X-100 for 15 min, and stained with Hoechst 33342 (Sigma, MO, USA) for 15 min. The cells were observed under a Leica DMIRB fluorescence microscope (objective 100×), as described previously [16 ].

The ATP content of calli generated from mature rice seeds was measured with a Luciferin-luciferase ATP Assay Kit (Beyotime, Shanghai, China). To analyse pollen viability, mature anthers from wild type and mutant plants were immersed into 1% (w/v) iodine and potassium iodide (I₂–KI) solution at room temperature with three biological replicates. The stained pollen grains were visualized and photographed under a Leica DMIRB fluorescence microscope.

Colocalization assays were performed to confirm the interaction between RGV-63R and RGV-91R. EPCs or GSTCs transfected with plasmid pEGFP-63R or pDsRed2-91R alone or cotransfected with both plasmids were fixed, permeabilized, stained with Hoechst 33342, and observed under a Leica DM IRB fluorescence microscope (objective 100×), as described previously [31 (link)].

Immunofluorescence analysis on SH‐SY5Y cells was performed as previously described 54. Briefly, after fixation with 4% Paraformaldehyde (PFA), cells were permeabilized in 0.2% Triton X100 and blocked by incubation with 10% normal goat serum (NGS, Gibco, Invitrogen, Milan, Italy) for 1 hr at room temperature (RT). The primary incubation was performed, overnight at 4°C, with the following antibodies: rabbit anti‐p75 (1:500, Chemicon Int. Inc., USA), mouse anti‐S‐100 (1:100; Sigma‐Aldrich, Saint Louis, Missouri, USA), rabbit anti‐HIF1A (1:200; Sigma‐Aldrich, Saint Louis, Missouri, USA), mouse anti‐Cx43 (1:150, Cell Signaling, Danvers, Massachusetts, USA) and rabbit β‐tubulin (1:200, Cell Signaling, Danvers, Massachusetts, USA).
After washing, slides were incubated with the appropriate secondary antibodies: fluorescence isothiocyanate (FITC) labelled anti‐rabbit antibody (1:200, Chemicon, Int. Inc., USA) and Cy3 labelled antimouse antibody (1:1000 Chemicon, Int. Inc., USA) for 1 hr at RT. Nuclei were stained with DAPI (1:1000) for 5 min. Finally, slides were mounted in fluorescent mounting medium Permafluor (Thermo Scientific, Wilmington, USA) and digital images were acquired using a Leica DM IRB fluorescence microscope and with Leica TCS SP8 confocal microscope. Nonspecific staining of cells was observed in control incubations in which the primary antibodies were omitted.

On the fifth day after BoNT/A injection, the three groups of rats (n = 3/group) were deeply anesthetized. Then, they were perfused with 0.9% saline and 4% paraformaldehyde (pH 7.4) in sequence. The left DRG was removed and placed in perfusion fixative (4°C) for 24 h. After fixation, paraffin embedding was performed, and 8-μm-thick sections were cut along the long axis of the DRG and mounted on 3-aminopropyl-triethoxysilane-coated glass slide. Sections were sealed with phosphate-buffered saline (PBS) containing 3% donkey serum albumin and 0.3% Triton X-100, and then incubated overnight with primary antibody, which was mouse anti-rat cl-SNAP-25 (1:500, MyBioSource, United States). After washing with PBS, DRG sections were incubated with donkey anti-mouse IgG (1:1,000 dilution Invitrogen) labeled with fluorescein isothiocyanate at 37°C for 40 min to determine the immunoreactivity of Cl-SNAP-25. Images were captured on a Leica DMIRB fluorescence microscope at a magnification of 20x. The immunofluorescent staining of protein expression was quantified and analyzed by ImageJ.