Preparation of tissue sample for ultrastructurel study (embedding in epoxy resin)
Preparation of Reynold’s lead citrate
Plunge freezing in liquid ethane
Making of glass knives
Instruments:
Balanced-break glass knife maker Leica EM KMR3
Description:
Glass knives are used for making of ultra-thin (about 70 nm) and semi-thin (up to 2000 nm) sections.
Reagents and supplies
Glass strip
The protocol of glass knives making
- 1. Wash the glass strip with ordinary soap and tap water. Dry it with clean towel. Don’t rub it intensively to prevent static charge, just blot it to dry.
- 2. After washing you hold the glass strip only at long factory-made facet or use gloves.
- 3. Align the glass strip with the precision click stop in knifemaker.
- 4. Simply lower the breaking head with the lever to its defined clamp position.
- 5. Push the button to perform an accurate score. Turn the breaking wheel to the position 5 for the strip 8 mm thick and 4 for 6 mm. After the break, the mechanisms automatically resets to the start position and ready for the next score.
- 6. After glass breaks, pull the drawer and put one half of glass aside.
- 7. Repeat steps 3-6 until you obtain two square fragment. One of them don’t rotate and put aside, the other rotate 45 degrees clockwise. Remember about holding the factory-made facet. Don’t touch fresh cleavage (to prevent water bridges during sectioning with this knife) or the upper side of glass (to prevent diamond contamination).
- 8. Push the The square corners are to be inserted in the pits on the drawner and under the breaking head.
- 9. Lower the breaking head with the lever, cut the glass and turn breaking wheel to the corresponding position (step 5).
- 10. After breaking the glass pull the drawer and take the knives at their sides. Don’t touch the back side of the knife. Cutting edges of two knives are on the opposite sides. The breaking arch goes right-down from left side at the cutting edge. The shoulder of good knife should not be more than 2 mm.
References:
Manufacture’s web site
Tutorial video
Preparation of tissue sample for ultrastructurel study (embedding in epoxy resin)
Instruments
Thermostatic oven Binder BD53
Magnet mixer BioSan MSH-300
Shaker BioSan Multi Bio 3D
Minitocker-shaker BioSan MR-1
Samplers for 2-20 mkl, 20-200 mkl, 100-1000 mkl
Description of the method:
The embedding of biological material in epoxy resin makes possible its studying with light and electron microscopes. 500-1500 nm sections are used for light microscopy, 60-80 nm sections – for transmission electron microscopy. The biological sample must be dehydrated before embedding in resin. Epoxy resins are usually not used for immulolocalization.
Reagents and supplies
Fixative solution (usually glutar aldehyde or the mix of glutar and paraform aldehyde)
Osmium tetraoxide
Buffer solution
Ethanol
Acetone
Epoxy resin
Pasteur pipets
Eppendorf tubes or vials
Protocol of sample preparation.
- 1. Pieces of tissue of organ put them in pre-cooled buffered fixative solution then cut cubes 1-4 mm3 with sharp blade. Fix the samples for 1-16 hours depending on the material and temperature of fixation.
- 2. Wash the samples with the same buffer 3-4 times for 5-15 min. Washed samples can be stored at +4 no more than 2 weeks.
- 3. Post fix the samples in 0,5-2% OsO4 (in the same buffer or water) for 45 min - 2 h at room temperature at +4 or overnight at +4 depending on the material.
- 4. Wash 3-4 times for 5-15 min. Washed samples can be stored at +4 for 1-2 days in buffer solution.
- 5. Material can be stained en block with 0.5-2.5% uranyl acetate in water for 30 min after though washing with water in 70% ethanol for 30-90 min during dehydration.
- 6. Dehydrate samples in the graded series of ethanol (30%), 50%, 70%, 95% for 5-10 minutes in each concentration, ethanol:acetone 1:1 1-2 times for 5-10 minutes, dry acetone 2 times for 5-10 minutes. Dehydration and embedding may be performed with gentle agitation on the plate of BioSanMultiBio 3D or BioSanMR-1 shakers.
- 7. For embedding in resin incubate material in graded series of resin in acetone (10%), 30%, 50%, 70%, 100%, increasing incubation time from 15-30 min to 1-4 h in each step. The sample can be left for overnight in 100% resin.
- 8. Plant material is often dehydrated in acetone only (30-50-70%, then 3 times of 100% acetone)
- 9. Propylene oxide can be used after of instead of acetone according to redcomendations of resin manufacture.
- 10. Fill embedding molds with the fresh resin and put the samples there. Incebate overnight at 45° and polymerize blacks according the manufacturer.
Reference:
J. A. Mascorro and J. J. Bozzola. Processing Biological Tissues for Ultrastructural Study // Electron microscopy : methods and protocols. — 2nd ed. / edited by John Kuo. p. 19-34.
Instruments
Precision balance Ohaus Adventurer Pro OH-AV413C
Description:
The staining of section on grids makes visible some elements of cell ultrastructure in transmission electron microscope. Reynold’s method of lead citrate preparation (Reynolds, 1963) gives the stable solution that can be stored for months.
Reagents and supplies
lead nitrate * 3H2O
sodium citrate
sodium hydroxide
volumetric flask 50 ml
pasteur pipettes
Preparation of Reynold’s lead citrate
- 1. Boil water to remove carbon dioxide than cool to about 40°
lead nitrate * 3H2O and 1.33 g , sodium citrate 1.76 g put in volumetric flask 50 ml, miz dry, add 30ml of warm water (from step 1) and shake for 30 min.
- 2. Add 8 ml of freshly made 1M NaOH. Solution in volumetric flask will clarify.
- 3. Add water (from step 1) to 50ml.
- 4. Store in refrigerator.
References:
Reynolds E. S. “The use of lead citrate at high ph as an electron-opaque stain in electron microscopy // The Journal of Cell Biology. 1963. V. 17 No 1 P. 208–212.
E.A. Ellis. Poststaining Grids for Transmission Electron Microscopy: Conventional and Alternative Protocols // Electron microscopy : methods and protocols. — 2nd ed. / edited by John Kuo. p. 97-106.
Plunge freezing in liquid ethane
Instruments
Leica EM GP
Description
Immersion freezing in liquid ethane is one of freezing techniques for watered samples that are designed to minimize water ice crystals that destroy the sample structure. This method is used in Resource Center together with other freezing methods: high pressure freezing and immersion freezing in iced (supercooled) liquid nitrogen.
Reagents and supplies
Compressed ethane
Liquid nitrogen
Coated grids
Filter paper
Tips for samplers
The protocol for freezing technique
- 1. Fill Leica EM GP with liquid nitrogen, fill camera with ethane and let it condense at liquid nitrogen temperature.
- 2. Prepare 1.2 mkl of solution under study to application on the grid.
- 3. Push LoadForceps. Place the coated grid in special forceps, fix the forceps at plunge bar.
- 4. Uncover the camera with liquid nitrogen, push LowerChamber.
- 5. Open the side window, apply the solution under study on one side of the grid.
- 6. Push Blot. Filter paper will blot the opposite side of the grid.
- 7. Push Plunge.
- 8. Push Transfer.
- 9. Take the forceps from the bar.
- 10. Place the grid in round grid box, screw the cover.
- 11. Store grid box in cryo-storage in liquid nitrogen.
- 12. For microscopy: place the round box in Gatan 914 camera filled with liquid nitrogen, move grid to microscope holder, close the shutter to prevent water condensation.
Referehce
Official manual of Leica Microsystems
(Leica EM GP.pdf)
