TITLE: Preparation of tissue for transmission electron microscopy ultrastructural analysis
AUTHORS: Jillian  C Danne, Simon A Crawford, Rachel Templin, Joan Clark, Viola Oorschot, Georg Ramm

[DESCRIPTION]
A transmission electron microscope (TEM) enables the magnification and visualisation of cell and tissue ultrastructure that cannot otherwise be resolved using a light microscope. In transmission electron microscopy, a high energy electron beam generated from an electron gun is focused by electromagnetic lenses onto an ultrathin section of plastic embedded specimen. Electrons are transmitted through the specimen onto a fluorescent screen based on sample density, and an image is acquired by a digital camera. Here, a protocol for preparing tissue specimens for TEM ultrastructural analysis is described. A tissue specimen is fixed, dehydrated and infiltrated with resin. The resin is then polymerised, and embedded tissue sectioned using an ultramicrotome. Ultrathin sections are contrasted with heavy metals prior to imaging using a TEM. Options for faster processing using a Microwave processor are described for relevant steps.

[STEPS]
SECTION: Fixation
1. All fixation and processing steps must be performed in a fume hood wearing appropriate personal protective equipment (PPE). The Material Safety Data Sheet (MSDS) for each chemical must be read before commencing.

Dissect out the tissue of interest on a Teflon plate or dental wax sheet using fine forceps and a scalpel blade at room temperature, and keeping the tissue submerged in Karnovsky's fixative, 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). Tissue pieces should be no larger than 1 mm cubed to ensure sufficient infiltration of fixative, solvents and resin.  
SECTION: Fixation
2. Place the tissue pieces in 5 ml tubes, 15 ml Falcon® tubes or a well-plate containing 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer and fix overnight at 4 degrees Celsius or for 2-4 hours at room temperature, on a rotor. 
SECTION: Fixation
3. Wash with 0.1M cacodylate buffer, 3 x 10 minutes at room temperature, agitating. 
SECTION: Fixation
4. Osmium tetroxide is extremely toxic and must be handled with care in a fume hood using the appropriate PPE. Used osmium should be discarded in a labelled plastic container containing ethanol to reduce and neutralise the osmium.

Osmium can precipitate out of solution when in the presence of glutaraldehyde to form small electron dense artefactual deposits. Samples must be washed thoroughly (Step 3) prior to post-fixation (Step 4) to avoid this. 

Potassium ferrocyanide reduces osmium, turning the post fixation solution black. Reduced osmium may also prevent the formation of artefactual precipitates. Potassium ferricyanide can be used as an alternative in combination with osmium tetroxide. This solution is a clear, amber colour.

The following steps can be performed with or without the use of a PELCO BioWave® Pro+ Microwave Processing System. A Biowave facilitates infiltration of reagents into tissue and cells, and reduces the processing time. 


Post fix the tissue in 1% Osmium tetroxide, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 2 hours at room temperature, agitating. Tube or well-plate lids must be well-secured during fixation. 

Biowave settings: 3x 120 seconds, 100W, Vacuum on. 
A two minute pause step without a solution change must be included in between each interval to prevent overheating of samples. 

SECTION: Fixation
5. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Washed samples can be stored at 4 degrees Celsius for up to 1 week until further processing.
SECTION: Dehydration and resin infiltration
6. For Steps 6-19, do not proceed if samples float following ethanol, acetone or Epon resin infiltration. 
Well-infiltrated samples should sink in solution. If they do not, samples may require extended infiltration times. 

Epon resin can be stored in the freezer but must come to room temperature before use. 


Dehydrate in 30% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.

SECTION: Dehydration and resin infiltration
7. Dehydrate in 50% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
8. Dehydrate in 70% ethanol in MilliQ water for 1 hour at room temperature, agitating. Tissue can be left at 4 degrees Celsius, overnight if necessary.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
9. Dehydrate in 90% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
10. Dehydrate in 95% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
11. Dehydrate in 100% ethanol for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
12. Dehydrate in 100% anhydrous ethanol for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the ethanol stock solution to dehydrate the solvent prior to use. 

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
13. Dehydrate in 100% acetone for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.

Propylene oxide can be used as an alternative to acetone (Steps 13-17), but is extremely toxic and may dehydrate samples too intensively, causing ultrastructural damage. 
SECTION: Dehydration and resin infiltration
14. Dehydrate in 100% anhydrous acetone for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the acetone stock solution to dehydrate the solvent prior to use. 

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
15. Optionally Infiltrate with 100% anhydrous acetone : epon resin (3 : 1 ratio) overnight at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
16. Infiltrate with 100% anhydrous acetone : epon resin (1 : 1 ratio) for 6 hours at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
17. Infiltrate with 100% anhydrous acetone : epon resin (1 : 3 ratio) overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on. 
SECTION: Dehydration and resin infiltration
18. Infiltrate with 100% epon resin for 6 hours at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
19. Infiltrate with 100% epon resin overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.

Residual acetone can adversely impact resin infiltration and polymerisation. A third 100% resin infiltration step may  be required to prevent this.
SECTION: Dehydration and resin infiltration
20. Transfer samples to labelled plastic embedding moulds, flat bottom BEEM® capsules or standard tip BEEM® capsules containing 100% epon resin. Polymerise in an oven set to 60 degrees Celsius for 24-48 hours. 
SECTION: Semi and ultrathin sectioning
21. Mount the resin-embedded tissue block in an ultramicrotome chuck, and secure the chuck to a stereo microscope fitted with a chuck mount. Manually trim the front face and all four edges of the tissue block into a trapezoid shape using a double edge razor blade. 
SECTION: Semi and ultrathin sectioning
22. Transfer the chuck and resin-embedded tissue block to a Leica UC7 ultramicrotome and secure in place. Trim the front surface of the block face until a full tissue face has been obtained. Trim manually using an 0.5 μm feed and a 45 degree glass knife (thickness 6.4 mm or 8 mm) or suitable diamond knife, for example a Diatome 35 degree angle ultra semi diamond knife. 
SECTION: Semi and ultrathin sectioning
23. To check the region of interest:  
SECTION: Semi and ultrathin sectioning
23.1. Cut 500 nm semi thin sections at a speed of 1 mm/sec using a Leica UC7 ultramicrotome and 45 degree glass knife  fitted with a filtered water-filled boat or suitable diamond knife. 
SECTION: Semi and ultrathin sectioning
23.2. Retrieve sections with an eyelash or Dalmatian hair mounted on a wooden stick and float sections in a drop of MilliQ water on a slide before drying on a mini hot plate. 
SECTION: Semi and ultrathin sectioning
23.3. Stain sections with Toluidine blue solution for 10-20 seconds on a mini hot plate before rinsing with MilliQ water and drying on a mini hot plate.

Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin tissue sections.
SECTION: Semi and ultrathin sectioning
23.4. Observe sections with a wide-field light microscope. If the region of interest has not been obtained, trim deeper and repeat Step 23. 
SECTION: Semi and ultrathin sectioning
24. Cut 90 nm gold coloured ultrathin sections at 1 mm/sec using a Leica UC7 ultramicrotome and a Diatome 45 degree angle ultra wet diamond knife. Spread the sections using a Max Wax heat pen to remove wrinkles. 
SECTION: Semi and ultrathin sectioning
25. Pick up sections on 50-200 mesh copper or palladium-coated copper grids using an eyelash or Dalmatian hair mounted on a wooden stick. Place grids section side up on a sheet of filter paper, cover with a glass petri dish and leave to dry for 10 minutes. Store grids in an enclosed grid box until use.
SECTION: Contrasting ultrathin sections
26. Place a sheet of parafilm on a clean flat bench or glass board using a small amount of water underneath to keep the film flat. 
SECTION: Contrasting ultrathin sections
27. For the following steps, use fine forceps or a perfect loop to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for MilliQ water, and 50-100 μl for uranyl acetate and lead citrate.

Stain sections with 0.6% filtered uranyl acetate in MilliQ water for 3 minutes.

Uranyl acetate enhances contrast of sections by interacting with proteins and lipids. The solution must be wrapped in foil and stored in a dark place to prevent precipitation from exposure to UV light.
SECTION: Contrasting ultrathin sections
28. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
29. Stain sections with 0.1-0.4% filtered lead citrate in MilliQ water for 3 minutes. 

Four drops of 10M sodium hydroxide should be added to the lead citrate stock solution prior to use to prevent lead citrate precipitating to form water-insoluble lead carbonate by exposure to carbon dioxide. 

Lead citrate enhances contrast of sections by interacting with proteins and glycogens, and binding to osmium and uranyl acetate.
SECTION: Contrasting ultrathin sections
30. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
31. Place grids section side up on a sheet of filter paper, cover with a glass petri dish and dry for 10 minutes. Store grids in a grid storage box.
SECTION: Contrasting ultrathin sections
32. 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.