Following the conclusion
of a pulse-chase experiment, cEND-coated
translucent PCIs were rinsed two times with PBS and then fixed for
10 min in 4% paraformaldehyde (VWR). Following three further washes
with PBS, the PCI membranes were carefully removed using a scalpel
blade and mounted vertically in 6% gum tragacanth (Sigma) on a 20
mm cork disc (Thermo Fisher Scientific). The mounted PCI was snap-frozen
in dry-ice-cooled isopentane and immediately stored at −80
°C. Using a CryoStar NX70 (Thermo Scientific) cryostat set to −20
°C, eight-micron cryosections were cut and mounted on Thermofrost
Plus (Thermo Fisher Scientific) glass cover slides. Individual sections
were isolated with a wax pen, rinsed three times with Tris-buffered
solution (TBS), and permeabilized for 10 min at room temperature with
0.1% Triton X-100/TBS. Sections were rinsed in 0.05% Tween 20 (wash
buffer—diluted in TBS) three times, followed by a 30 min block
incubation at room temperature in 10% bovine serum albumin (BSA—diluted
in TBS). Sections were rinsed in wash buffer three times, followed
by an overnight incubation at 4 °C with primary antibodies diluted
in 10% BSA/TBS (1/100 rat antitransferrin receptor (NovusBio), 1/50
goat anti-CD31 (R&D Systems), or 1/100 rabbit anti-Rab5 (Abcam)).
Sections were rinsed with wash buffer three times, followed by 1 h
room temperature incubation with 1/500 host-targeted fluorescently
labeled antibodies (goat antirat Alexa 488, donkey antigoat Alexa
488, goat antirabbit Alexa 555, or donkey antimouse 555 (Thermo Fisher
Scientific)). Sections were rinsed with wash buffer three times and
mounted with a glass coverslip in Fluoromount-G medium (Thermo Fisher
Scientific) supplemented with 100 ng/mL 4′,6-diamidino-2-phenylindole
(DAPI). Epifluorescent images were taken using a 10× objective
[numerical aperture (NA) 0.30] by an Olympus BX53 fluorescent microscope
(Mercury Vapor Short Arc lamp). DAPI was detected using excitation
bandwidths 360/370 nm and emission bandwidths 420/460 nm. Alexa 488
was detected using excitation bandwidths 470/495 nm and emission bandwidths
510/550 nm. TIFF images were taken using cellSens Dimension software
and further prepared for publication using ImageJ software. Confocal
images were taken using a 63× oil objective (NA 1.4–85
nm pixel size) by an LSM700 microscope with Zeiss Zen software. Lasers
DAPI 405 (wavelength ranges 420–480 nm), 488 (wavelength ranges
< 572 nm), and 555 (wavelength range greater than 560 nm) were
detected using photomultiplier tubes (PMTs) using pinhole sizes 60,
69, and 65 μM, respectively. Images were further processed using
deconvolution software Huygens Professional (signal-to-noise ratio
30—Scientific Volume Imaging). ImageJ software was used to
create an audio video interleave (AVI) movie from individual deconvoluted z-stack confocal LSM5 images and also to display Alexa 488
images (TfR) with a false-color look-up table (LUT) to display intensity
levels on the membrane cell surfaces.
of a pulse-chase experiment, cEND-coated
translucent PCIs were rinsed two times with PBS and then fixed for
10 min in 4% paraformaldehyde (VWR). Following three further washes
with PBS, the PCI membranes were carefully removed using a scalpel
blade and mounted vertically in 6% gum tragacanth (Sigma) on a 20
mm cork disc (Thermo Fisher Scientific). The mounted PCI was snap-frozen
in dry-ice-cooled isopentane and immediately stored at −80
°C. Using a CryoStar NX70 (Thermo Scientific) cryostat set to −20
°C, eight-micron cryosections were cut and mounted on Thermofrost
Plus (Thermo Fisher Scientific) glass cover slides. Individual sections
were isolated with a wax pen, rinsed three times with Tris-buffered
solution (TBS), and permeabilized for 10 min at room temperature with
0.1% Triton X-100/TBS. Sections were rinsed in 0.05% Tween 20 (wash
buffer—diluted in TBS) three times, followed by a 30 min block
incubation at room temperature in 10% bovine serum albumin (BSA—diluted
in TBS). Sections were rinsed in wash buffer three times, followed
by an overnight incubation at 4 °C with primary antibodies diluted
in 10% BSA/TBS (1/100 rat antitransferrin receptor (NovusBio), 1/50
goat anti-CD31 (R&D Systems), or 1/100 rabbit anti-Rab5 (Abcam)).
Sections were rinsed with wash buffer three times, followed by 1 h
room temperature incubation with 1/500 host-targeted fluorescently
labeled antibodies (goat antirat Alexa 488, donkey antigoat Alexa
488, goat antirabbit Alexa 555, or donkey antimouse 555 (Thermo Fisher
Scientific)). Sections were rinsed with wash buffer three times and
mounted with a glass coverslip in Fluoromount-G medium (Thermo Fisher
Scientific) supplemented with 100 ng/mL 4′,6-diamidino-2-phenylindole
(DAPI). Epifluorescent images were taken using a 10× objective
[numerical aperture (NA) 0.30] by an Olympus BX53 fluorescent microscope
(Mercury Vapor Short Arc lamp). DAPI was detected using excitation
bandwidths 360/370 nm and emission bandwidths 420/460 nm. Alexa 488
was detected using excitation bandwidths 470/495 nm and emission bandwidths
510/550 nm. TIFF images were taken using cellSens Dimension software
and further prepared for publication using ImageJ software. Confocal
images were taken using a 63× oil objective (NA 1.4–85
nm pixel size) by an LSM700 microscope with Zeiss Zen software. Lasers
DAPI 405 (wavelength ranges 420–480 nm), 488 (wavelength ranges
< 572 nm), and 555 (wavelength range greater than 560 nm) were
detected using photomultiplier tubes (PMTs) using pinhole sizes 60,
69, and 65 μM, respectively. Images were further processed using
deconvolution software Huygens Professional (signal-to-noise ratio
30—Scientific Volume Imaging). ImageJ software was used to
create an audio video interleave (AVI) movie from individual deconvoluted z-stack confocal LSM5 images and also to display Alexa 488
images (TfR) with a false-color look-up table (LUT) to display intensity
levels on the membrane cell surfaces.
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