C hgf microscope

Gene localization of the mogrol biosynthesis pathway was observed in strain CEN-PK2-1C. Strain CEN-PK2-1C was transformed by plasmids pY15-SgCDS-GFP, pY15-SgEPH3-GFP, pY15-CYP87D18-GFP-mCherry-SEC12, and pY15-AtCPR1-GFP-mCherry-SEC12, thereby yielding strains LMOR001, LMOR002, LMOR003 and LMOR004. These strains were streaked onto an SD-Leu plate and cultured at 30°C for 2 days. Later, a single colony was picked into a 2 ml SD-Leu medium, cultured at 30°C and 220 rpm for 24 h, and washed twice with 1× phosphate-buffered saline (0.8% NaCl, 0.02% KCl, 0.144% Na₂HPO₄, and 0.024% KH₂PO₄, pH 7.4). Subsequently, 2-μL preparations were directly plated on slides. Strain LMOR001 culture was stained with Nile red solution in acetone (1:10, v/v; 1 mg/ml; Solarbio) and incubated for 60 min in the dark at 25°C to further confirm the specific distribution of SgCDS.
Fluorescence imaging was performed on a Nikon C-HGF microscope with a ×100 oil immersion objective. GFP fluorescence (excitation, 490 nm; emission, 530 nm), mCherry fluorescence (excitation, 580 nm; emission, 615 nm), and Nile red fluorescence (excitation, 561 nm; emission, 615 nm) were detected by microscopy. Image analysis was carried out on the Leica TCS SP8 software package and ImageJ (NIH).

For FM4-64 staining, strains were incubated with 8 µM FM4-64 for 5 min in culture medium. For di-4-ANEPPDHQ staining, concentrated yeast strains were incubated in 5 μM di-4-ANEPPDHQ solution (diluted with YPD medium) for 10 min on ice and then washed at least 3 times with cold YPD medium. The 488-nm laser was used to excite the samples, and the detection ranges of the two channels were set at 500–580 and 620–750 nm, respectively.
Generalized polarization (GP) images and GP values were generated and calculated according to the previously published methods [27 (link), 42 (link)]. Upon multiplying the GP value by the intensity value of each pixel, the hue-saturation-brightness (HSB) value can be generated. For FM4-64 and di-4-ANEPPDHQ staining, a Leica TCS SP8 microscope fitted with a 60 × oil-immersion objective was used. For PI staining, concentrated yeast strains were incubated in PBS (pH 6.0) at 30 °C for 10 min. Fluorescence imaging was performed under a Nikon C-HGF microscope fitted with a 100 × oil immersion objective. Apoptotic cells stained with PI were observed using a TRITC fluorescent filter.