700 confocal microscope

Bodipy or other immunofluorescent staining was used to evaluate the lipid accumulation or gene expression, respectively. 8 µm frozen liver sections were fixed in 10% NBF for 10 min then washed with PBS. Sections were then blocked for 20 min with 10% normal goat serum diluted in PBS, followed by a rinse in PBS. Thereafter, sections were incubated with bodipy dye for 30 min or with primary antibody overnight in a 4 °C incubator. The next day, specimens were washed with PBS and stained with the secondary antibody for 45 min in the dark at room temperature. Finally, tissues were mounted with DAPI mounting medium and stored in −20 °C until analyzed with a Zeiss 700 confocal microscope. All images were taken at 20× magnification and quantified using Fiji ImageJ software. Detailed antibody information is listed in Table 3.

For cytosolic pH measurements, 4-day-old UB10:pH-GFP plants43 (link) were transferred to 2 ml of liquid ½MS (pH 5.5, 2.15 g l⁻¹ MS salts, 0.5 g l⁻¹ MES) for 30 min with the indicated drugs or mock (DMSO). Imaging was done with a 700 confocal microscope (Zeiss) with a Plan-Apochromat 20 × /0.8 M27 objective lens. pH-GFP was excited by 405 and 488 nm diode lasers and the emission was collected separately between 500 and 555 nm. The images were evaluated in ImageJ (Fiji) by measuring intensities of both channels in six circulars ROIs per root meristematic zone. Vacuolar pH was measured as described before44 (link) for a 2-h incubation with the indicated small molecules.

Approximately 120,000 HUVEC cells stably expressing EV or FLAG-K1 were plated in MatTek 35 mm glass-bottom dishes. Cells were washed with PBS and fixed by 15-minute incubation in 3.7% formaldehyde at room temperature. Cells were washed 3X with PBS and then permeabilized by 15-minute incubation at room temperature in 0.2% Triton-X 100/PBS. Cells were then washed again 3X with PBS and then blocked in 10% bovine serum albumin (BSA) PBS for 30 minutes. Cells were stained 1:200 with a directly conjugated FITC-ECS (DDDDK) polyclonal antibody (Bethyl laboratories) and anti-AMPKβ1/2 (1:50, Cell Signaling) in 10% BSA for 1 hour at room temperature. Cells were washed 2X quick followed by 3X 5-minute washes. Samples were then incubated with anti-rabbit Alexa Fluor 647 (1:600) secondary antibody in 10% BSA at room temperature for 1 hour. All samples were then stained with DAPI for 1 minute and washed. Fluorescent images were acquired by taking z-stacks using a 63X oil objective on a Zeiss 700 confocal microscope. The overlap coefficient according to Manders (R) was determined using Image J. Confocal images from 5 cells in their entirety were evaluated for co-localization by calculating the overlap coefficient.

Tissue Sections (4 µm thick) were deparaffinized with xylene and hydrated with a decreasing ethanol gradient (100%, 95%, 70%, 50%, dd-H₂O), after which antigen recovery was performed by heating in a water bath at 95 °C in the citrate buffer (10 mM sodium citrate pH 6.0, 0.05% Tween-20) for 15 min. After allowing sections to cool for 20 min, sections were washed with TBST 3 times for 5 min each and blocked with 5% goat serum. Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum. After, sections were washed with TBST and incubated with anti-mouse/anti-rabbit secondary antibodies conjugated with Alexa 488 and Alexa 694, respectively, for 1 h at room temperature. Then, samples were washed and incubated with DAPI for 10 min and covered with Prolong Gold Antifade Mounting Media (Invitrogen, p36930, Waltham, MA, USA). Images were acquired with a Zeiss 700 confocal microscope equipped with a 40 × objective. All image processing was done using Zen Blue ver. 3.6.

Primary isolations were performed on three resistant (104A, 3A, 18A) and one susceptible (96B) self-derived progeny of ‘Ma851’ to investigate the presence or absence of GFP-Foc-STR4. Fungal strains were isolated 3 months post-inoculation with GFP-Foc-STR4 infected millet. Five regions were isolated per plant, which included the upper stem just below the first leaf petiole, mid-point of the stem, stem just above the rhizome, the central cylinder of the rhizome and the outer layer of the rhizome connected to the cortex. Four pieces of tissues were isolated in each region. These sectioned pieces were surface sterilized for 1 min in 1% hypochlorite solution, then rinsed twice in distilled water for 30 s each time. Each piece was air-dried and plated onto water agar plates containing 100 mg L^-1 streptomycin for 5 days at 24°C. Segments showing Fusarium-like growth under a dissecting microscope were further isolated and then hyphal tipped to generate monoconidial isolates on half strength potato dextrose agar (PDA) plates containing 50 mg per L hygromycin B. Each isolate was then transferred to 20 mL of potato dextrose broth containing 50 mg L^-1 hygromycin B (PDB) and incubated at 26°C on a platform rotating at 160 rpm for 4 days. GFP fluorescence of the isolates was visualized under Zeiss 700 confocal microscope to confirm the presence of the GFP-Foc-STR4 strain.

Immunohistochemical staining for AR-V7 was done on serial 4 μm breast tissue sections as described previously [62 (link)] using the AR-V7 EPR15656 antibody (1:200), biotinylated anti-rabbit antibody (1:400, DAKO Corp., Carpinteria, CA), streptavidin-horseradish peroxidase complex (1:500, DAKO), and diaminobenzidine tetrahydrochloride. To analyze 22Rv1 cells (Supplementary Figure S4), a standard cytospin protocol was utilized [63 (link)] followed by immunohistochemistry as above.
Preparation of formalin-fixed, paraffin-embedded tissue sections for immunofluorescence was done as described previously [64 (link)]. For all antigens, retrieval was performed in 600 mL of 10 mM Tris base and 1 mM Na-EDTA (pH 9.0) by heating in a 1100W microwave at full power for 5 min and subsequently heating at 50% power for an additional 5 min. Primary antibodies used for immunofluorescence were AR-441 (M3562, 1:50, DAKO) and AR-V7 (EPR15656, 1:400). Primary antibodies were detected using secondary antibodies conjugated to either Alexa-Fluor 488 (A11029; Life Technologies) or Alex-Fluor 568 (A11036; Life Technologies). Images were acquired sequentially on a Zeiss 700 confocal microscope with a pinhole aperture of 2 airy units.