Metodika přípravy vzorků
Vážený/á Uživateli/ko, tohle jsou vzorové formy metodik, podle kterých můžeš napsat tu svoji do svého vědeckého článku. Měj prosím na paměti, že tvoje metodika se v určitých bodech bude lišit, a proto ji uprav podle svých poznámek o přípravě vzorku, nebo konzultuj s lidmi, kteří ti vzorek pro pozorování pomáhali připravit, nejlépe bude asi kontaktovat: holland@paru.cas.cz
Ve vzorových metodikách jsou některá slova zvýrazněná tučně, je to proto, že v těchto bodech se může metodika lišit s velmi vysokou pravděpodobností. Může se jednat o jiný přístroj, jiný typ chemikálie, atd.
Pod každou metodikou je uvedený článek, který bys měl uvést jako původní protokol, který jsi pro svůj vzorek upravil.
Chemická fixace
Cells… were fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer, washed three times 15 minutes in same buffer at pH 7.4. Specimen was post-fixed in 1 % osmium tetroxide in cacodylate buffer for 2 hours at room temperature, dehydrated in a graded series of ethanol (30, 50, 70, 80, 90, 95 and 100 % each for 15 minutes) and infiltrated with Spurr resin, SPI-pon (SPI), acetone: ratios of 1:2, 1:1 and 2:1 each for 1 hour and overnight in pure embedding media under vacuum condition using desiccator. Polymerization was done in 48 hours at 62 °C.
Sections were prepared using a Ultracut UCT (Leica) microtome and collected on 300/400 mesh copper grids (SPI). Staining was performed using alcoholic uranic acetate for 30 min and in lead citrate for 20 min. Images were obtained using a 1400 Flash (JEOL) transmission electron microscope.
Adapted from: DOI: 10.1093/molbev/msad134
HPF/FS
CELLS were high-pressure frozen using EM ICE (Leica) high-pressure freezer. Freeze substitution EM AFS2 (Leica) was carried out in 2% osmium tetroxide diluted in 100% acetone at -90 °C for 16 h, and then warmed up at a rate of 5 °C per hour to remain at -20 °C for 14 h, and finally warmed up again at the same rate to a final temperature of 4 °C. Samples were rinsed three times in anhydrous acetone at room temperature and infiltrated stepwise in acetone mixed with SPI-pon resin (SPI) (acetone : SPI ratios of 2 : 1, 1 : 1 and 1 : 2, for 1 h at each step). The samples, now in pure resin, were polymerized at 60 uC for 48 h.
Sections were prepared using a Ultracut UCT (Leica) microtome and collected on 300/400 mesh copper grids (SPI). Staining was performed using alcoholic uranic acetate for 30 min and in lead citrate for 20 min. Images were obtained using a 1400 Flash (JEOL) transmission electron microscope.
Adapted from: DOI: 10.14411/fp.2014.023
Tomography:
For electron tomography, protein A-conjugated 10 nm gold nanoparticles (Aurion) were added to one/both side/s of each section as fiducial markers. Finally, the section/s surface/s was/were carbon coated. Tilt series images were collected in the range of ±60° with 1° increments using a 200 kV 2100F (JEOL) transmission electron microscope equipped with a high-tilt stage and direct detector K2 (Gatan) and controlled by SerialEM automated acquisition software (Mastronarde, 2005). Images were aligned using the fiducial markers. Electron tomograms were reconstructed using the IMOD software package. Manual masking of the area of interest was employed to generate a 3D surface model (Kremer et al., 1996).
Adapted from: https://doi.org/10.1016/j.mito.2022.11.006
Tokuyasu:
After 24 h of treatment with 2 µM hexyl-TPP or vehicle, Cells were fixed in 4 % formaldehyde with 2.5 % glutaraldehyde in 0.1 M HEPES. After 1 h incubation at RT and at 4 °C overnight, cells were washed with 0.1 M HEPES and cryoprotected in 2.3 M sucrose for 72 h at 4 °C and frozen by plunging into liquid nitrogen. Ultrathin sections (100 nm) were cut at -100 °C, picked-up with 1.15M sucrose/1 % methylcellulose solution (25 cp, Sigma). Sections were incubated with gold nanoparticles (15 nm) for 3 min, washed in dH2O, and contrasted/embedded using a mixture of 2 % methylcellulose and 3 % aq. uranyl acetate solution (9:1) for 10 min. Samples were observed with a MegaView III camera mounted on a JEOL 1010 TEM.
Adapted from: https://doi.org/10.1016/j.mito.2022.11.006
Cryo-EM
Samples were deposited on glow discharged (Solarus II Plasma Cleaner, GATAN) holey carbon grids and rapidly frozen in liquid ethane with plunge freezer (LEICA EM GP2, Leica Microsystems, Germany) and visualized at 200 kV Jeol JEM-2100F TEM (JEOL, Tokyo, Japan). Images were recorded using GATAN K2 direct detection camera. Image analysis was carried out in RELION 3.0.
Adapted from: https://doi.org/10.1016/j.bbabio.2022.148946
Negative Staining
Cells were fixed in 2.5% glutaradehyde overnight, washed in 0.1M HEPES buffer and adhered to the surface of formwar-carbon coated, glow discharged TEM grids. The cells were then stained with 1% aq. uranyl acetate for 30s. Th exces of the solution was removed by blotting paper and the grids were let to dry.
Adapted from: https://doi.org/10.1093/pcp/pcaa148
RT-SEM:
Cells were fixed in 2.5% (v/v) glutaraldehyde in 0.1M phosphate buffer pH 7.2 and transferred to the poly-L-lysine coated cover slips, post fixed with 2% OsO4 in 0.1 M phosphate buffer for 2 hours, dehydrated in acending acetone series, critical point drying with CO2 in Pelco CPD2 (Ted Pella Inc. Redding, CA, USA) and sputter coatd with gold in a Sputter Coater Polaron chamber (Polaron Ltd., Watford, UK). The cells were then observed in JEOL 7401-F (JEOL Europe,Prague, Czech Republic).
Adapted from: DOI: 10.14411/fp.2014.023
Cryo SEM
Sample was rapidly frozen in the slushy nitrogen created in CryoALTO slushing chamber (CryoALTO 2500, Gatan, Inc.) and inserted under vacuum to a FESEM JEOL 7401F (JEOL Ltd.) equipped by the cryo attachement CryoALTO. The sample was kept intact in the CryoALTO pre-chamber tor 10 minutes to remove the ice contamination artifact by a sublimation at -98°C to -95°C under a high vacuum. Then sputter-coated with a layer of gold (10-20 s) in the pre-chamber, which is equipped with a cold sputter coater. The ample was then inseted into the EM chamber and the topography was observed using the Everhart-Thornley detector a 1-3 kV, at -140°C.
Adapted from: DOI: 10.3389/fmicb.2017.00596
SBF-SEM
The sample preparation by the high-pressure freezing technique followed the protocol for TEM sample preparation. After freeze-substitution, the samples were subsequently stained with 1% thiocarbohydrazide in 100% acetone for 1.5 h, 2% OsO4 in 100% acetone for 2 h at room temperature, and 1% uranyl acetate in 100% acetone overnight at 4°C. After every staining step, the samples were washed 3 times with 100% acetone for 15 min. Samples were then infiltrated with 25%, 50%, or 75% acetone-resin mixture for 2 h at each step, and finally infiltrated in 100% Hard Resin Plus 812 (EMS) overnight and polymerized at 62°C for 48 h. Resin-embedded blocks were trimmed and imaged using an Apreo SEM equipped with a VolumeScope (Thermo Fisher Scientific, Germany). Serial images were acquired at 3.5 keV, 50 pA, 40 Pa with a resolution of 6 nm, 100-nm slice thickness, and dwell time per pixel of 4 μs.
Adapted from: DOI: 10.1128/mbio.03279-22
Microarray tomography
Cell pellets were transferred to specimen carriers and immediately frozen in the presence of 20% wt/vol bovine serum albumin solution using a high-pressure freezer Leica EM ICE (Leica Microsystems). Freeze substitution was performed in the presence of 2% OsO4 diluted in 100% acetone at −90°C for 96 h. Specimens were warmed to −20°C at a step of 5 °C/h and, subsequently, to 3°C at a 3°C/h step. At RT, samples were washed in acetone and infiltrated with 25%, 50%, 75% acetone/resin mixture for 1 h at each step. Finally, samples were infiltrated in 100% Epon resin, Embed-812 (EMS), and polymerized at 60°C for 48 h. Ultrathin (70 nm) sections were collected on a silicon wafer, which was glued to the SEM pins using double carbon tape, post-stained with uranyl acetate for 45 min and lead citrate for 20 min, and carbon coated. Array sections were imaged using Apreo SEM and MAPS software (Thermo Fisher). Serial images were acquired using immersion mode: accelerating voltage 2.5 keV, probe current 0.2 nA, working distance 5.6 mm, resolution 4 nm, slice thickness 70 nm, dwell time per pixel 3 µs.
Adapted from: https://doi.org/10.1128/mbio.01921-23
