We cleaned 18-mm #1.5 coverslips (Warner Instruments) with either 1% hydrofluoric acid (HF) for 5 min or 1% Hellmanex II (Fisher) for 3 h, followed by distilled water and 100% ethanol. Cleaned coverslips were then flamed and placed in sterile 35-mm tissue culture dishes. For PALM, cells were grown on fibronectin coated (2 μg ml−1 in PBS (pH 7.4); Sigma) cover-slips and transiently transfected 48 h after plating with Fugene 6 (Roche). Approximately 24 h after transfection, cells were washed twice with PBS, fixed with 4% paraformaldehyde, 0.2% glutaraldehyde (Electron Microscopy Sciences) in PBS for 35 min at room temperature (25 °C) (or for 15 min at 4 °C followed by 30 min at room temperature for cells incubated at 4 °C). Quenching was done with filter-sterilized 10 mg ml−1 BSA in PBS for 5 min, and cells were finally washed four times with PBS. In all cases, fixation was preformed just before the imaging. Cells expressing VSVG were incubated at 32 °C at least 8 h before fixation. To ensure high density of TfR-PAGFP on the plasma membrane, cells were incubated with 100 μM deferoxamine mesylate salt (DFO, Sigma) for 18 h after transfection. Coverslips were incubated with 1:4,000 diluted Tetraspec beads (Invitrogen) in PBS for 10 min that served as fiducial markers.
For confocal microscopy, cells were grown on coverslips (18-mm, #1.5) and transfected 24 h before imaging.
To generate GPMVs, COS-7 cells transiently expressed with mEGFP-tagged proteins were labeled with 200 μg ml−1 Rh-DOPE (Avanti Polar Lipids) dissolved in ethanol for 5 min, washed twice with GPMV buffer (2 mM CaCl2, 10 mM Hepes and 150 mM NaCl, pH 7.4), and incubated with freshly prepared GPMV active reagent consisting of 2 mM N-ethyl maleimide (Sigma) in GPMV buffer for 1 h at 37 °C with shaking (60 cycles min−1). GPMVs were then gently decanted into a tube and allowed to sit undisturbed on ice for 30 min to allow the larger GPMVs to sediment. Finally, GPMVs were collected from the bottom 20% of the total volume of the tube and imaged at 25 °C.
To covalently immobilize on coverslips, clean coverslips were first treated with 5% (wt/vol) 3-aminopropyl trimethoxysilane (APTMS) in acetone for 15 min at room temperature, washed with acetone and PBS in succession, and then incubated with 0.25% (wt/vol) glutaraldehyde in PBS for 30 min. PAGFP in PBS was centrifuged (100,000g in TLA 45 rotor for 2 h) to minimize any potential aggregation before the experiment. Next, functionalized coverslips were incubated with a combination of 10 nM PAGFP and 5 μM BSA in PBS for 30 min at room temperature in a humid chamber. Coverslips were extensively washed after the incubation and then imaged in PBS.
For confocal microscopy, cells were grown on coverslips (18-mm, #1.5) and transfected 24 h before imaging.
To generate GPMVs, COS-7 cells transiently expressed with mEGFP-tagged proteins were labeled with 200 μg ml−1 Rh-DOPE (Avanti Polar Lipids) dissolved in ethanol for 5 min, washed twice with GPMV buffer (2 mM CaCl2, 10 mM Hepes and 150 mM NaCl, pH 7.4), and incubated with freshly prepared GPMV active reagent consisting of 2 mM N-ethyl maleimide (Sigma) in GPMV buffer for 1 h at 37 °C with shaking (60 cycles min−1). GPMVs were then gently decanted into a tube and allowed to sit undisturbed on ice for 30 min to allow the larger GPMVs to sediment. Finally, GPMVs were collected from the bottom 20% of the total volume of the tube and imaged at 25 °C.
To covalently immobilize on coverslips, clean coverslips were first treated with 5% (wt/vol) 3-aminopropyl trimethoxysilane (APTMS) in acetone for 15 min at room temperature, washed with acetone and PBS in succession, and then incubated with 0.25% (wt/vol) glutaraldehyde in PBS for 30 min. PAGFP in PBS was centrifuged (100,000g in TLA 45 rotor for 2 h) to minimize any potential aggregation before the experiment. Next, functionalized coverslips were incubated with a combination of 10 nM PAGFP and 5 μM BSA in PBS for 30 min at room temperature in a humid chamber. Coverslips were extensively washed after the incubation and then imaged in PBS.
