TITLE: Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy
AUTHORS: Rotimi Fasimoye, Alan Prescott, Dario R Alessi

[DESCRIPTION]
Transmission electron microscopy (TEM) is a tool used to image, in good resolution, the structure of organelles within the cell. Available protocols are designed to image structures within fixed intact cells. Here, we described a protocol where Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) (as described in ddx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.

[STEPS]
SECTION: Method
1. Perform GolgiTAG-IP as previously described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1
SECTION: Method
2. After last wash of beads with KPBS, fix the beads in 1 mL solution containing 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 Molarity (M) sodium cacodylate buffer dilute in water for 60 min at Room temperature.

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3. Pellet beads with magnet DynaMagTM Spin Magnet (or equivalent) and remove the supernatant.
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4. Wash beads pellet three times in 1 mL 0.1 Molarity (M) sodium cacodylate buffer, pelleting the beads with magnet after each wash. 

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5. After third wash, spin down beads in tabletop centrifuge at 1000 x g for 1 min. Pellet should be tightly packed.

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6. Use a needle to gently dislodge the pellet and use a Pasteur pipette to remove the pellet to a clean glass vial. (Note: All steps are carried out in a glass vial).
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7. To ensure rapid dehydration and embedding, cut pellet with scalpel into approximately 1mm3 pieces,  then post-fix in 1 mL 1% (w/v) Osmium tetroxide with 1.5% (w/v) sodium ferrocyanide in 0.1 Molarity (M) sodium cacodylate buffer for 60 min at Room temperature.

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8. Wash pellets three times in  0.1 Molarity (M) sodium cacodylate buffer, by adding the buffer, allowing the pellets to settle and gently taking out supernatant with P1000 tip.
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9. After the third wash, wash with water three times (as in step 8), then  resuspend in 1% w/v tannic acid and 1% w/v uranyl acetate diluted in water for  60 min . Remove supernatant.


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10. Dehydrate pellet through alcohol series: 
50% ethanol for 10 min
70% ethanol for 10 min
80% ethanol for 10 min
90% ethanol for 10 min
95% ethanol for 10 min
then twice in 100% ethanol for 10 min (1/2)
100% ethanol for 10 min (2/2)
 


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11. Further dehydrate in 100% propylene oxide twice for 10 min.

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12. Resuspend pellet into 50% v/v propylene oxide and 50% v/v Durcupan resin 10 min.

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13. Open glass vial to allow propylene oxide to evaporate (This will take approximately 60 min).

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14. To embed pellet in resin, resuspend in 100% Durcupan resin.
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15. Embed polymerise resin in an oven at 60 °C for 2880 min.

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16. Cut sections of polymerised embedded resin with Leica UCT ultramicrotome.
 

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17. Stain sections with 3% aqueous uranyl acetate for 20 min.

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18. Stain with Reynold lead citrate for 20 min.

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19. Image stained sections on JEOL 1200EX TEM using SIS III camera.
 
  

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8.1. Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 1 min  (1/3).
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8.3. Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 1 min  (3/3).
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8.2. Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 1 min (2/3).
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11.1. Further dehydrate in 100% propylene oxide twice for 10 min (1/2).
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11.2. Further dehydrate in 100% propylene oxide twice for 10 min (2/2).