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Transmission Electron Microscopy
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 Materials Required

 

1. Mammalian cells
2. Phosphate buffered Saline
3. 2.5% glutaraldehyde.
4. 0.1M Sodium cacodylate buffer pH 7.4
5. 1% osmium tetroxide
6. 1% uranyl acetate
7. 50%, 70%, 80%, 90%, and 100% ethanol
8. Amyl acetate
9. 100% acetone
10. Infiltrating media (1:1 acetone/polyBed 82)
11. Flat Mold
12. Epoxy resin
13. Oven
14. Microtome
15. CO2 incubator
16. Petriplates
17. Suction pump
18. Inverted microscope
19. Transmission electron microscope
20. Refrigerator
21. Centrifuge


Procedure

 

  1. Prepare a petriplate containing cells cultured in DMEM media.
  2. Take out a petriplate containing the cells from the CO2 incubator. Check the growth of the cells under a phase contrast microscope. Take the petriplate to the fume hood.
  3. Suction out the media from the petriplates using a suction pump inside the fumehood.

    Fixation:
     
  4. Wash the petriplate with 2ml of 0.1 M sodium cacodylate buffer (pH=7.4) two times carefully.
  5. Remove the sodium cacodylate buffer and add 2ml of 2.5% glutaraldehyde solution at room temperature into the cells. Keep it for 1 hour to fix the cells.  
  6. Discard the gluteraldehyde solution and wash the fixed cells three times with 0.1M sodium cacodylate buffer (pH 7.4).
  7. Discard the sodium cacodylate buffer and add 2ml of 1% osmium tetroxide solution to the petriplate and incubate for 1hour at 4 degree Celsius.
  8. Discard the osmium tetroxide solution and wash 3 times with 0.1M sodium cacodylate buffer.
  9. Discard the buffer and add 2ml of filtered 1% uranyl acetate to the petriplates containing cells and incubate for 20 minutes at 4 degree Celsius.
  10. Discard the uranyl acetate solution and wash 3 times with 2ml of 0.1M sodium cacodylate buffer
  11. Discard the sodium cacodylate buffer.

    Dehydration:
     
  12. Take ethanol in series of concentrations: 50%, 70%, 80%, 90% and 100% ethanol (% v/v. Add 2ml of 50% ethanol into the petriplate containing the fixed cells and incubate for 5 minutes.
  13. Discard the 50% ethanol and then incubate the cells with 2ml of 70% ethanol for 5 minutes. After 5 minutes discard the 70% ethanol.
  14. Add 2ml of 90% ethanol to the cells and 10 minute incubation. Discard the 90% ethanol after 10 minutes. Then add 2 ml of 100% ethanol and incubate for 10 minutes. Discard the ethanol after 10 minutes.
  15. Add 2ml of amyl acetate into the petriplate. Gently swirl the petriplate for uniform mixing of the amyl acetate solution. Then take 1ml of the solution from the petriplate and transfer it into a 1.5ml microfuge tube.
  16. Centrifuge the microfuge tube at 1000 rpm for 1 min and collect the cell pellet.
  17. Add 1ml of Amyl acetate to the microfuge tubes and remove amyl acetate after a quick centrifuge at 1000 rpm and remove the amyl acetate.  Repeat it 5 times (each time 1minute incubation.
  18. Centrifuge at 1000 rpm for 1 min and discard the amyl acetate and collect the cell pellet.
  19. Wash with 1ml of 100% Acetone and incubate for 2minutes at room temperature.
  20. Centrifuge at 1000 rpm for 1 min throw the acetone and collect the cell pellet. Repeat this washing step two more times.
  21. Add 1ml of infiltrating media [1:1 acetone /PolyBed 812] to the pellet and keep it for 1 hour incubation at room temperature.

    Embedding:
     
  22. Take flat molds. Transfer the infiltrated cells from the microfuge tube to the mold and add epoxy resin to the cells.
  23. Keep in oven at 60 degree Celsius for allowing the resin to set overnight.

    Sectioning:
     
  24. After the resin has set, cool the blocks containing cells and then cut using diamond knife into thin sections in a microtome.
  25. Transfer the thin sections into a copper grid for viewing under the transmission electron microscope.  

 

 

 

 

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