Experimental chambers were assembled by sandwiching two coverslips using parafilm. Before use, the chambers were passivated with a β-casein solution at a concentration of 5 mg/ml in PBS for at least 5 min at RT. GUVs were incubated with 0.2 μM BLOC-1 in the experimental chambers for at least 15 min at RT before observation. To mix GUVs with BLOC-1, we first added 8 μl of the GUVs in 15 μl of an “outer buffer” (60 mM NaCl, 20 mM Tris, pH 7.5), followed by adding 2 μl of BLOC-1 (2.5 μM in stock). Samples were observed using a spinning disk confocal Nikon eclipse Ti-E microscope equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon) and a CMOS camera, Prime 95B (Photometrics). After formation of tubules, sample was pipetted off and flash-frozen for cryo-EM experiments.
Cfi plan apo vc objective
Manufactured by Nikon
Sourced in Japan
The Nikon 100× CFI Plan Apo VC objective is a high-magnification microscope objective designed for laboratory applications. It features a numerical aperture of 1.45 and a working distance of 0.13 mm. The objective utilizes Nikon's Chromatic-Free Infinity (CFI) optical system and Plan Apo lens design to provide high-resolution, accurate images.
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4 protocols using cfi plan apo vc objective
1
BLOC-1 Induces Membrane Tubulation in GUVs
2
Reconstitution of PI(4,5)P2-Enriched GUVs for Membrane Binding Studies
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For all experiments, GUVs composed of brain total lipid extract (36 ) supplemented with 5 mole percentage (mol%) brain PI(4,5)P2, 0.2 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], and 0.5 mol% BODIPY-TR ceramide were prepared by electroformation on platinum electrodes overnight at 4°C in a physiologically relevant salt buffer. The salt buffer outside GUVs was 20 mM Tris (pH 7.5), 60 mM NaCl, and 100 mM sucrose. The salt buffer inside GUVs was 20 mM Tris (pH 7.5), 60 mM NaCl, and 100 mM glucose.
GUVs were incubated with IRSp53 I-BAR domain at a bulk concentration of 0.02–0.1 μM for at least 30 min at room temperature before observation. For all experiments, microscope slides and coverslips were washed with water and ethanol followed by passivation with a β-casein solution at a concentration of 5 mg/mL for at least 5 min at room temperature. GUVs were observed by Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon), and QuantEM:512SC camera (Photometrics, Tucson, AZ).
GUVs were incubated with IRSp53 I-BAR domain at a bulk concentration of 0.02–0.1 μM for at least 30 min at room temperature before observation. For all experiments, microscope slides and coverslips were washed with water and ethanol followed by passivation with a β-casein solution at a concentration of 5 mg/mL for at least 5 min at room temperature. GUVs were observed by Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon), and QuantEM:512SC camera (Photometrics, Tucson, AZ).
3
Gag and I-BAR Membrane Recruitment and Tubulation
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GUVs were first incubated with either Gag or I-BAR domain at bulk concentrations depending on the designed experiments for at least 15 min at room temperature before adding either I-BAR domain or Gag, respectively, into the GUV-protein mixture. In experiments where there was only Gag but no I-BAR domain, the stock solution of I-BAR domain was used in order to obtain a comparable salt strength outside GUVs as those where I-BAR domain was present. The GUV-protein mixture was then incubated for at least 15 min at room temperature before observation. For the Gag/I-BAR membrane recruitment assay, samples were observed on a Nikon C1 confocal microscope equipped with a 60× water immersion objective (Nikon, CFI Plan Apo IR 60× WI ON 1.27 DT 0.17). For the Gag/I-BAR tubulation assay, samples were observed with an inverted spinning disk confocal microscope Nikon eclipse Ti-E, equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon) and a CMOS camera, Prime 95B (Photometrics).
For all experiments, coverslips were passivated with a β-casein solution at a concentration of 5 g.L−1 for at least 5 min at room temperature. Experimental chambers were assembled by placing a silicon open chamber on a coverslip.
For all experiments, coverslips were passivated with a β-casein solution at a concentration of 5 g.L−1 for at least 5 min at room temperature. Experimental chambers were assembled by placing a silicon open chamber on a coverslip.
4
Biofilm Visualization via CLSM Imaging
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For visualisation of static biofilms with confocal laser scanning microscopy (CLSM), stained coverslips were rinsed with PBS and inverted onto a PBS-filled rubber frame secured on a microscope slide. Imaging was performed using a Nikon A1R confocal laser scanning microscope fitted with CFI PLAN APO VC objective (Nikon 60x/1.40 Oil). Images were captured with NIS-Elements C (v4.4, Nikon) software and processed using Imaris (v8.2, Bitplane) software.
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