TITLE: 2D TEM CLEM (Correlative Light Microscopy and Electron Microscopy)
AUTHORS: Pietro De Camilli, Yumei Wu

[DESCRIPTION]
This protocol details the general procedure of Correlative Light Microscopy and Electron Microscopy (CLEM) with conventional chemical fixation and 2D Transmission Electron Microscopy (TEM) imaging.

[STEPS]
SECTION: General preparation:
1. Culture cells on poly-d-lysine-coated 35 mm MatTek dish (P35G-1.5-14-CGRD) and transfect the cells with the plasmids of the interest.
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2. Pre-fix the cells in 37 °C-warmed 4% PFA and 0.25% glutaraldehyde in the imaging buffer.

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3. Wash in the same imaging buffer.

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4. Image with a fluorescence microscopy, find the regions of the interest and their coordinates on the dish, then take the Z-stack fluorescence images and the grids map/coordinate by using phase contrast.
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5. Fix the cells in 2.5% glutaraldehyde and 0.1 Molarity (M) sodium cacodylate buffer for 60 min.

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6. Wash in 0.1 Molarity (M) sodium cacodylate buffer.

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7. Fix in 2% OsO4 and 1.5% K4Fe(CN)6 (Sigma-Aldrich) in 0.1 Molarity (M) sodium cacodylate buffer for 60 min.

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8. Wash in MiliQ water.

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9. En bloc stained with 2% aqueous uranyl acetate 5 min at 4 °C.
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10. Wash in MiliQ water.
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11. Dehydration in 50% etoh for 5 min.
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12. Dehydration in 75% etoh for 5 min.

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13. Dehydration in 95% etoh for 5 min.
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14. Dehydration in 100% etoh.

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15. Infiltration in 50% Epon for 0.5-1 hour.
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16. Infiltration in pure Epon 812.

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17. Add two drops of pure Epon into the center of the glass bottom of the culture dish.
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18. Polymerize at 60 °C for 1-2 days.

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19. Cell of interest were relocated based on the pre-recorded coordinates under a stereomicroscope.
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20. Trim and cut ultrathin sections (50-60 nm) with microtome.
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21. Observe the ultrathin sections in a electron microscope.
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3.1. Wash 5 min in the same imaging buffer. (1/3)
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3.2. Wash 5 min in the same imaging buffer. (2/3)
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3.3. Wash 5 min in the same imaging buffer. (3/3)
SECTION: General preparation:
6.1. Wash in 0.1 Molarity (M) sodium cacodylate buffer for 5 min. (1/4)
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6.2. Wash in 0.1 Molarity (M) sodium cacodylate buffer for 5 min. (2/4)
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6.3. Wash in 0.1 Molarity (M) sodium cacodylate buffer for 5 min. (3/4)
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6.4. Wash in 0.1 Molarity (M) sodium cacodylate buffer for 5 min. (4/4)
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8.1. Wash in MiliQ water for 5 min. (1/4)
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8.2. Wash in MiliQ water for 5 min. (2/4)
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8.3. Wash in MiliQ water for 5 min. (3/4)
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8.4. Wash in MiliQ water for 5 min. (4/4)
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10.1. Wash in MiliQ water for 5 min. (1/4)
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10.2. Wash in MiliQ water for 5 min. (2/4)
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10.3. Wash in MiliQ water for 5 min. (3/4)
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10.4. Wash in MiliQ water for 5 min. (4/4)
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14.1. Dehydration in 100% etoh for 10 min . (1/3)
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14.2. Dehydration in 100% etoh for 10 min . (2/3)
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14.3. Dehydration in 100% etoh for 10 min . (3/3)
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16.1. Infiltration in pure Epon 812 for 60 min. (1/2)
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16.2. Infiltration in pure Epon 812 for 60 min. (2/2)