Oct 27, 2022
Tokuyasu processing and immuno-electron microscopy of cells
- Jillian C Danne1,
- Viola Oorschot2,
- Georg Ramm1
- 1Ramaciotti Centre for Cryo EM, Monash University, Melbourne, Australia;
- 2Electron Microscopy Core Facility, EMBL, Heidelberg, Germany
Protocol Citation: Jillian C Danne, Viola Oorschot, Georg Ramm 2022. Tokuyasu processing and immuno-electron microscopy of cells. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7nr6v5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2022
Last Modified: October 27, 2022
Protocol Integer ID: 63175
Keywords: Tokuyasu, Cryosectioning, Immunolabelling, Protein-A-gold, Antibodies, Cells, Ultrastructure, Transmission electron microscope, cryopreservation, electron microscopy of cell, using cryo, electron microscopy technique, electron microscopy, microscopy technique, transmission electron microscopy imaging, thawed cryo, tokuyasu processing, protocol for the tokuyasu technique, transmission electron microscope, freezing sample, cryo, immunolabelling of subcellular structure, processing of cell, microscopy imaging, antigenicity of sample, tokuyasu technique, antigenicity, cell, immunolabelling, immuno, ultrastructural morphological information, tissue into plastic
Abstract
Ultrastructural morphological information is routinely obtained from the processing of cells and tissue into plastics for transmission electron microscopy imaging. This technique involves the use of strong chemical fixatives, solvents and resins, which destroy the antigenicity of samples. Freezing samples to a vitreous state using cryo-electron microscopy techniques preserves ultrastructure without the use of harsh chemicals, however, immunolabelling cannot be achieved at temperatures as low as -196 degrees Celsius. Here we describe a protocol for the Tokuyasu technique for cryopreservation and immunolabelling of subcellular structures in cells. Cells are lightly chemically fixed and cryoprotected in sucrose before freezing in liquid nitrogen. Cells are sectioned by cryo-ultramicrotomy and retrieved cryo-sections are thawed. Thawed cryo-sections are then immunolabelled, stained and imaged using a transmission electron microscope.
Guidelines
Liquid Nitrogen is extremely cold (-196 degrees Celsius) and can cause severe burns. Personal Protective Equipment (PPE) must be worn when handling liquid nitrogen.
The following chemicals must be handled with extreme care in a fume hood using the appropriate PPE:
Uranyl acetate is mildly radioactive and extremely toxic if ingested, inhaled or in contact with abraded or cut skin.
Paraformaldehyde and glutaraldehyde are toxic, corrosive and potentially carcinogenic.
Materials
Paraformaldehyde, 16% (wt/vol)Electron Microscopy SciencesCatalog #15710
Glutaraldehyde 8% (wt/vol)Polysciences, Inc.Catalog #00216
0.1M Phosphate buffer pH 7.4
0.2M Phosphate buffer pH 7.4
1x Phosphate buffered saline pH 7.4
GlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G-7403
Gelatin from porcine skinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G1890-500g
SucroseMerck Millipore (EMD Millipore)Catalog #1.07654.1000
Ice
Liquid nitrogen
Methylene blueMerck MilliporeSigma (Sigma-Aldrich)Catalog #115943
Azure IIMerck MilliporeSigma (Sigma-Aldrich)Catalog #861065
Toluidine blue Merck MilliporeSigma (Sigma-Aldrich)
MilliQ Water
Methyl cellulose Merck MilliporeSigma (Sigma-Aldrich)Catalog #M-6385
Formvar powder ProSciTechCatalog #C064
Bovine serum albumin, heat shock fraction pH7Merck MilliporeSigma (Sigma-Aldrich)Catalog #A9647
Mouse anti-glutamine synthetase antibodyMerck Millipore (EMD Millipore)Catalog #MAB302
Rabbit anti-mouse Ig antibodyRocklandCatalog #610-4120
Protein A-Gold 10nmUMC UtrechtCatalog #PAG 10nm
2% Uranyloxylate pH 7.0
Uranyl acetateElectron Microscopy SciencesCatalog #22400
Equipment
Petri dish 100mm x 20mm
NAME
Greiner Bio-One
BRAND
664160
SKU
Equipment
BRAND® Petri dish, glass 150mm x 25mm
NAME
BRAND®
BRAND
BR455751
SKU
Equipment
CELLTREAT Scientific Products 60mm x 15mm Petri Dish
NAME
CELLTREAT
BRAND
50-828-744
SKU
Equipment
Parafilm M
NAME
Bemis
BRAND
IA041
SKU
Equipment
3ml graduated transfer pipette
NAME
Copan
BRAND
200C
SKU
Equipment
Dry block heater
NAME
Ratek
BRAND
DBH4000D
SKU
Equipment
Corning cell scraper
NAME
Corning
BRAND
CLS3011
SKU
Equipment
Paper towel
NAME
Tork
BRAND
2187951
SKU
Equipment
BRAND disposable microcentrifuge tubes, 1.5mL with safety lid
NAME
Brand
BRAND
BR780400-450EA
SKU
Equipment
Sprout plus mini centrifuge
NAME
Sprout
BRAND
120610
SKU
Equipment
Transfer pipette, standard bulb, PE, fine tip, capacity 5ml
NAME
ProSciTech
BRAND
LCH192
SKU
Equipment
Block heater
NAME
Ratek
BRAND
DBH20D
SKU
Equipment
Oven MINO/6/CLAD
NAME
Genlab
BRAND
N/A
SKU
Equipment
Single edge carbon steel blade
NAME
Electron Microscopy Sciences
BRAND
71960
SKU
Equipment
SafeSeal Microcentrifuge Tube
NAME
2 mL
TYPE
Sarstedt
BRAND
72.695.500
SKU
LINK
Equipment
Fine Forceps
NAME
Forceps
TYPE
Dumont
BRAND
11251-10
SKU
LINK
Equipment
Black teflon plate
NAME
N/A
BRAND
N/A
SKU
Equipment
Glass petri dish, 100mm
NAME
BRAND
BRAND
BR455751
SKU
Equipment
Blade scalpel ST #11
NAME
Swann Morton
BRAND
21016SM
SKU
Equipment
Double edge blades
NAME
Personna
BRAND
72000
SKU
Equipment
Rotary tube mixer
NAME
Ratek Instruments
BRAND
RSM7DC
SKU
Equipment
Sample pin for cryo-ultramicrotomes, aluminium
NAME
Leica
BRAND
75959-06
SKU
Equipment
Filter paper, grade 1, 12.5cm
NAME
Whatman
BRAND
1001-125
SKU
Equipment
UC7/FC7 Cryo-ultramicrotome
NAME
Leica
BRAND
EMFC7
SKU
Equipment
Cryotrim20 diamond knife
NAME
Diatome
BRAND
TT-20
SKU
Equipment
Cryo immuno diamond knife, 3mm
NAME
Diatome
BRAND
DCIMM3530
SKU
Equipment
Stainless steel loop, 3mm
NAME
Contributed by user
BRAND
N/A
SKU
Equipment
Plain glass slides 76mm x 39mm x 1.0-1.2mm
NAME
Thermo Scientific
BRAND
AGL4222A
SKU
Equipment
Mini hot plate
NAME
Thermofisher
BRAND
HP2310BQ
SKU
Equipment
Olympus widefield microscope, model CHK2-F-GS
NAME
Olympus
BRAND
N/A
SKU
Equipment
Eyelash or Dalmatian hair mounted on a wooden stick
NAME
N/A
BRAND
N/A
SKU
Equipment
50 Mesh palladium/copper grids
NAME
Gilder
BRAND
GCU-PD50
SKU
Equipment
50 Mesh copper grids
NAME
Gilder
BRAND
GCU50
SKU
Equipment
150 Mesh palladium/copper grids
NAME
Gilder
BRAND
GCU-PD150
SKU
Equipment
150 Mesh copper grids
NAME
Gilder
BRAND
GCU150
SKU
Equipment
24-well clear flat bottom TC-treated multi-well cell culture plate
NAME
Falcon
BRAND
FAL353047
SKU
Equipment
Perfect loop
NAME
Diatome
BRAND
70944
SKU
Equipment
Foil
NAME
N/A
BRAND
N/A
SKU
Equipment
Remanium wire loop
NAME
N/A
BRAND
N/A
SKU
Equipment
Polystyrene esky
NAME
N/A
BRAND
N/A
SKU
Equipment
Leica EM ACE200 coater
NAME
Leica
BRAND
N/A
SKU
Equipment
Grid storage box, 100 grid capacity
NAME
Gilder
BRAND
HL065
SKU
Equipment
JEOL JEM-1400 Plus 120keV Transmission electron microscope
NAME
JEOL
BRAND
N/A
SKU
Troubleshooting
Fixation
All fixation steps must be performed in a fume hood wearing the appropriate personal protective equipment (PPE). The Material Safety Data Sheet (MSDS) for each chemical must be read before commencing.
Adherent cells grown in a petri dish must be submerged in solution at all times to prevent desiccation. Petri dishes should be clearly labelled on the bottom to prevent sample mix up.
Different fixatives can influence the antigenicity of cells and tissue. The following two fixation solutions are typically tested to determine the best immunogold labelling outcome:
2% Paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer:
Remove cells from the incubator and gently add a volume of double concentrate fixative, 4% paraformaldehyde, 0.4% glutaraldehyde in 0.1 M phosphate buffer (PB) equal to the volume of culture media in the petri dish. Ensure the fixative and culture media are well mixed, and incubate for 5 mins at room temperature.
4% Paraformaldehyde in 0.1 M phosphate buffer:
Remove cells from the incubator and gently add a volume of normal concentrate fixative, 4% paraformaldehyde in 0.1 M PB equal to the volume of culture media in the petri dish. Ensure the fixative and culture media are well mixed, and incubate for 5 mins at room temperature. Double strength fixative is not used with this fixation solution to avoid osmotic shock of cells.
Remove the fixative and refresh with normal strength fixative, either 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M PB or 4% paraformaldehyde in 0.1 M PB. Seal the petri dish with parafilm and fix cells overnight at 4 degrees Celsius.
Optionally, to store samples at 4 degrees Celsius for up to 1 month, remove the fixative and add 1% paraformaldehyde in 0.1 M PB. Place a sheet of parafilm directly over the petri dish, place the lid over the parafilm and seal the lid to the petri dish with more parafilm to prevent desiccation.
Gelatin embedding and sucrose infiltration
Remove the fixative and wash with phosphate buffered saline (PBS), 3 x 5 mins at room temperature.
Wash with 0.15% glycine in PBS for 10 mins.
Samples should be embedded in gelatin one at a time from Step 6.
Remove 0.15% glycine in PBS and add 1.5ml or 2.5 ml of 1% gelatin in 0.1 M PB (for 6 cm or 10 cm petri dishes, respectively) using a 3ml graduated transfer pipette. The gelatin should be pre-warmed to 37 degrees Celsius.
Gently detach adherent cells from the bottom of the petri dish using a cell scraper.
Transfer the cells to a 1.5 ml microcentrifuge tube using a 3 ml graduated transfer pipette, and pellet the cells immediately using a benchtop microcentrifuge.
Remove the supernatant using a 3 ml fine tip transfer pipette and place the closed microcentrifuge tube containing the cell pellet on a heat block set to 37 degrees Celsius.
Repeat Steps 6-9 for the remaining samples.
Add approximately 400 μL to 1000 μL of pre-warmed (37 degrees Celsius) 12% gelatin in 0.1 M PB to the microcentrifuge tube and gently resuspend the cell pellet using a 3 ml graduated transfer pipette. Ensure no air bubbles are present and immediately place the sample on a heat block or in an oven set to 37 degrees Celsius to infuse for 20 to 30 minutes.
Pellet cells using a benchtop microcentrifuge and remove the supernatant leaving a volume of 12% gelatin approximately twice that of the cell pellet (ie 100 μL of 12% gelatin for a 50 μL sized cell pellet).
Cells will be stacked perpendicular to the g-forces.
Solidify the 12% gelatin with cells by cooling at 4 degrees Celsius for 30 minutes.
12% gelatin provides support to cells so they are easier to handle and facilitates cryosectioning.
Steps 14-19 should ideally be performed in a cold room to prevent condensation of water, which can affect the osmolarity of 2.3 M sucrose in PBS. If a cold room is not available, ensure the sample and sucrose are kept cool using ice.
Cut the end off the microcentrifuge tube containing approximately 30 μl of gelatin embedded cells using a razor blade and immediately submerge this in a 2 ml microcentrifuge tube containing ice cold 2.3 M sucrose in PBS.
2.3 M sucrose in PBS shrinks 12% gelatin aiding separation of the embedded cell pellet from the plastic microcentrifuge tube.
Cut off another piece of the microcentrifuge tube containing the remaining gelatin embedded cell pellet using a razor blade and immediately submerge this in a 2 ml microcentrifuge tube containing ice cold 2.3 M sucrose in PBS.
Wait a few minutes and then carefully remove the gelatin embedded cell pellet from the microcentrifuge tube tip using fine forceps. If the cell pellet does not easily separate from the plastic, return it to the 2.3 M sucrose in PBS and repeat Step 16.
Place a Teflon plate on top of a glass petri dish and add a drop of 2.3 M sucrose in PBS to the Teflon plate. If a cold room is not available, cool the Teflon plate by inverting the glass petri dish on ice.
Place the gelatin embedded cell pellet dome-side up in the drop of ice cold 2.3 M sucrose in PBS and cut the dome twice down the middle into three segments using a scalpel or double edged razor blade. Cut the middle segment into strips and then cut each strip into cubes no larger than 1mm3.
Strips can be cut into rectangles no more than 1mm wide to identify the orientation of cells.
Using forceps, carefully place the gelatin-embedded cell cubes or rectangles into a 2 ml microcentrifuge tube containing ice cold 2.3 M sucrose in PBS, and infuse overnight at 4 degrees Celsius, rotating.
2.3M sucrose cryoprotects samples so they are vitreous when frozen. Samples shrink, dry and become brittle if left in sucrose for too long. Do not infiltrate cell blocks for more than 2 days.
Freezing
Mount each gelatin-embedded cell block on a clean aluminium bullseye pin. Orient the cell block flat and long side down using forceps, and remove the excess sucrose with a strip of filter paper so the block surface appears shiny and enough sucrose remains at the block base to cement it to the pin.
If the cell block is too big or too much sucrose is used for mounting, a freezing imbalance between the pin and sample can lead to the formation of cracks at the base of the cell block during freezing. Consequently, the block may break away from the pin during cryo-sectioning.
Blocks larger than 1mm3 should be mounted in the centre of the pin. If a block requires reorienting, add a drop of ice cold 2.3 M sucrose in PBS to the pin and repeat Step 20.
Freeze each sample block by gently plunging the pins in liquid nitrogen. For large blocks (>1mm3 ) or blocks with few cells (mostly gelatin), cool the sample pin in the cryo-ultramicrotome chamber at -100 degrees Celsius for 10 mins prior to freezing in liquid nitrogen. Pins should be mounted and frozen one at a time.
Frozen samples can be stored for years.
Cryosectioning
Trim the front face and edges of the frozen tissue block cutting 50-100 μm deep on all four sides. Cut at a speed of 100 mm/sec with a 100 nm feed, at -100 degrees Celsius using a Leica UC7/FC7 cryo-ultramicrotome and Diatome cryotrim 20 knife.
To check the region of interest:
Cut semi thin frozen sections with a feed of 100-300 nm, at 0.8 mm/sec and at -100 degrees Celsius using a Diatome cryo immuno diamond knife.
Pick up semi thin frozen sections in a 1:1 mixture of 2% methyl cellulose: 2.3 M sucrose in 0.1M PBS (pick-up solution) using a 3 mm stainless steel loop. For details on section retrieval, see Steps 26.1-26.3).
Transfer the sections to a glass slide by carefully pressing the loop containing the pick-up solution with sections onto the slide.
Stain the sections with methylene blue/Azure II or toluidine blue solution for 10-20 seconds on a mini hotplate before rinsing with milliQ water and drying on a mini hotplate.
Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin cell sections.
Observe sections with a wide-field light microscope. If the region of interest has not been obtained, continue trimming and repeat Step 23.
Cut a ribbon of approximately four 60-70 nm ultrathin frozen sections at 0.8 mm/sec and -90 to -120 degrees Celsius using a Leica UC7/FC7 cryo-ultramicrotome and Diatome cryo immuno diamond knife.
A short ribbon allows the sections to stretch in the pick-up solution once thawed, minimising the occurrence of section wrinkles.
The cutting temperature can be lowered for thinner sections or increased for compact or well-fixed samples. If the temperature is too high, the sample block can soften and sections may compress.
Carefully detach the ribbon of ultrathin frozen sections from the diamond knife edge and drag the ribbon away from the knife edge using an eyelash or Dalmatian hair mounted on a wooden stick (one stick in each hand).
Pick up the ultrathin frozen sections in a 1:1 mixture of 2% methyl cellulose : 2.3 M sucrose in PBS (pick-up solution) using a 3 mm stainless steel loop as follows:
Dip the stainless steel loop into a microcentrifuge tube containing the pick-up solution to create a thin film of solution over the loop. The droplet must not be too thick (large meniscus) or too thin (no meniscus).
Bring the stainless steel loop containing the pick-up solution into the cryochamber and towards the ribbon of sections. As soon as the pick-up solution at the edge of the loop starts to freeze white, carefully press the partially frozen droplet onto the ribbon of sections.
Take extreme care to avoid damaging the diamond knife edge with the stainless steel loop.
Retract the stainless steel loop containing the pick-up solution with sections from the cryochamber and wait for the droplet to thaw completely.
Transfer the sections to 50-150 mesh copper or palladium-coated copper grids with carbon-coated formvar film by carefully pressing the loop containing the pick-up solution with sections onto the grid.
During formvar film preparation, coated grids are secured to a sheet of parafilm on a glass slide.
Sample grids immersed in pick-up solution can be stored for several years in a sealed petri dish at 4 degrees Celsius until further use.
Immunogold labelling of ultrathin sections for transmission electron microscopy
Fill the wells of a 24-well plate with PBS and float grids section side down in the solution. Use a fine tip pasteur pipette to remove enough PBS from each well so that the lid can be secured on the well-plate without disturbing the floating grids. Seal the 24-well plate lid with parafilm.
Put the plate with the floating grids in an oven set to 37 degrees Celsius for 60 minutes to remove the 2% methyl cellulose, 2.3M sucrose and 12% gelatin from the cell sections.
Place a sheet of parafilm on the work bench using a small amount of water underneath to keep the film flat.
For the following steps, use a perfect loop or fine forceps to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for rinsing solutions and 5-10 μl drops for antibodies on the clean parafilm surface. Spin all antibodies and protein A-gold (PAG) for 30 seconds using a benchtop microcentrifuge prior to use.
Quench aldehydes with 0.15% glycine in PBS, 5 x 2 mins.
Block with 1% bovine serum albumin (BSA) in PBS for 5 mins.
Incubate with a primary antibody diluted in 1% BSA/PBS for 45 to 60 minutes in a dark moist chamber at room temperature. A chamber can be prepared by placing wet paper towel inside a foil-covered 15 cm glass petri dish. Place the antibody drops on a slide covered with parafilm.
Example: Mouse anti-glutamine synthetase (1:500 dilution), to label radial glial cells.
Rinse with 0.1% BSA in PBS, 5 x 2 mins.
Optionally incubate with a bridging antibody diluted in 1% BSA/PBS for 30 mins, in a dark moist chamber at room temperature.
Example: Rabbit anti-mouse Ig bridging antibody (1:1000 dilution).
Rinse with 0.1% BSA in PBS, 5 x 2mins.
Incubate with 10 nm Protein A Gold (PAG, dilution as recommended by manufacturer for specific batch) diluted in 1% BSA/PBS for 30 mins, in a dark moist chamber at room temperature.
Rinse with PBS, 5 x 2 mins.
Fix with 1% glutaraldehyde in PBS for 5 mins at room temperature to stabilise the immunogold labelling reaction. This step must be completed in the fume hood using the appropriate PPE.
Rinse with distilled water, 6 x 1 min.
If the glutaraldehyde is not rinsed out completely, it will react with uranyloxalate (Step 42) to form electron dense precipitates.
Stain the grid in a drop of 2% uranyloxalate (pH 7.0) for 5 mins at room temperature.
Cover a glass petri dish with parafilm using a small amount of water under the film to keep it flat. Place the petri dish on ice and add 3 drops of 2% methyl cellulose : 4% uranyl acetate (9 ml : 1 ml) (pH 4.0) to the surface.
Float the grid briefly in the first two drops of methyl cellulose/uranyl acetate, then transfer to the third drop and leave for 10 mins.
Remove and dry the grid using the looping out method:
Clean a remanium wire loop with water and dry.
Push the loop into the methyl cellulose/uranyl acetate drop and under the grid.
Lift the grid out from the methyl cellulose/uranyl acetate drop using the loop.
Tilt the loop and grid at a 45-60 degree angle and bring the loop to a piece of filter paper.
As soon as the loop contacts the filter paper, drag it slowly along the filter paper to remove the excess liquid. A thin film of methyl cellulose/uranyl acetate should remain on the surface of the grid. The slower the drag, the thinner the film.
Dry the grid suspended in the loop for 30 minutes at room temperature.
Remove the grid from the loop using fine forceps taking care not to tear the methyl cellulose/uranyl acetate film away from the grid. Store grids in a grid storage box.
Place a grid in a transmission electron microscope grid holder for high resolution imaging using a transmission electron microscope.
Example: JEOL JEM-1400 Plus TEM at 80 keV equipped with a high sensitivity bottom mount CMOS 'Flash' camera.