Freeze fracture replicas were produced using a previously described method (Tarusawa et al., 2009 (link)) with some modifications. Cells (2 × 105 cells/ml) were seeded onto polyethylene terephthalate filters with 0.4-µm pore size (#353090; Corning) and cultured for 6 d. The cells were washed with 0.1 M phosphate buffer (PB) and fixed with 2% glutaraldehyde in 0.1 M PB at 4°C overnight. After washing with 0.1 M PB, the samples were cryoprotected with 30% glycerol in 0.1 M PB at 4°C overnight, and then rapidly frozen in between two copper carriers using a high-pressure freezing machine (HPM010; BAL-TEC). The cells were then fractured by separation of the two carriers at −120°C and replicated by platinum (45° unidirectional from horizontal level, 2 nm thick) and carbon (20 nm thick) in a freeze-fracture replica machine (BAF060; BAL-TEC). The replicated materials were transferred to a solution containing kitchen bleach (50%) and incubated with shaking until cell debris was removed from the replicas. The replicas were washed twice with distilled water and picked up onto grids coated with Pioloform (Agar Scientific). The samples were observed with a JEM1010 transmission EM (JEOL) at 100 kV accelerating voltage. Images were captured with a Veleta CCD camera using iTEM software (Olympus Soft Imaging Solutions). All freeze-fracture images are presented apical-side up in the figures.
Jem1010 transmission em
Manufactured by JEOL
Sourced in Japan
The JEM1010 is a transmission electron microscope (TEM) produced by JEOL. It is designed for high-resolution imaging and analysis of small-scale specimens. The JEM1010 utilizes an electron beam to magnify and focus images of samples, allowing users to observe fine details and structures at the nanometer scale.
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4 protocols using jem1010 transmission em
1
Freeze Fracture Replica Preparation
2
Ultrastructural Analysis of HSV-1 Infection
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HOG-PLP cells were mock-infected or infected with HSV-1 at a m.o.i. of 100 and incubated for 1 h at 4°C. Then cells were washed with PBS and fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C for 2 hour. After that, cells were incubated overnight with 8% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C. Fixed cells were washed in PBS containing 20 mM glycine and embedded in 12% gelatine, infiltrated with 2.3 M sucrose and frozen in liquid nitrogen. Cryosections were stained with a mouse monoclonal anti-PLP antibody. Primary antibody was detected with 15 nm anti-mouse-gold (British BioCell, Cardiff, UK). Cryosections were examined with a JEM1010 transmission EM (Jeol, Tokyo, Japan).
3
Visualizing HSV-1 Infection in HOG Cells
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HOG cells cultured at 37°C in GM or DM were mock-infected or infected with HSV-1 at an m.o.i. of 50. At different time points post-infection, cells were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 37°C for 2 hours. Then, they were washed in PBS containing 20 mM glycine and processed by freeze substitution as previously described [13] (link). Samples were examined with a JEM 1010 transmission EM (Jeol, Tokyo, Japan).
4
Visualizing HSV-1 Effects on MAL Proteolipids
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HOG MAL-diHcRed cells were mock infected or infected with HSV-1 at an MOI of 1 and incubated for 24 h at 37°C. Then cells were washed with PBS and fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C for 2 h. After that, cells were incubated overnight with 8% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C. Fixed cells were washed in PBS containing 20 mM glycine and embedded in 12% gelatin, infiltrated with 2.3 M sucrose, and frozen in liquid nitrogen. Cryosections were stained with a rabbit anti-RCFP antibody, which detects diHcRed, the red tag attached to exogenous MAL proteolipids. Primary antibody was detected with 15 nm anti-rabbit-gold (British BioCell, Cardiff, UK). Cryosections were examined with a JEM1010 transmission EM (Jeol, Tokyo, Japan).
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