OTO Fixation for SEM and Blockface Imaging with Rehydration Step for UA and Lead
by Fabrice Richard and Nicolas Brouilly (Aggad et al., 2023)

Insprired by Hall, Hartweig and Nguyen and Deerink et al., 2002

Protocol

- Cool the freeze substitution (FS) device to -90°C

- Prepare the first FS solution: 2% OsO4 in acetone; and transfer it to the FS device

- Transfer the High Pressure frozen sample to the FS device and launch the program  
  90 to 106h at -90°C; ramp from -90°C to -30°C in 20h; hold -30°C for 16h; ramp from -30°C to 0°C in 10h

- Prepare the 1% TCH (Sigma 88535-5g) solution in acetone (preheat the required volume of acetone at 60°C in a tightly closed bottle without too much of dead volume, add the TCH, put back in the oven and stir every 20 min for 1-2h)

- Wash in acetone over 1h (4x15’) on ice

- Stain in 1% TCH in acetone for 60 min at RT (5 to 10% of the TCH powder may have not dissolved, take the supernatant)

- Wash 4X to 8X in acetone over 60 min (6x10’) at RT

- Stain in 2% OsO4 in acetone for 60 min at RT

- Wash in acetone over 1h (4x15’) at RT

- Rehydrate the sample in graded water series:
  10%, 20%, 40%, 60%, 80% and 100% H2O (15’ each, cold solutions but incubation at RT)

- Stain in 1% UAc in water ON at RT

- Prepare the lead aspartate solution such as in Deerinck et al. 2022

1. the day before, prepare the L-aspartic acid solution [0.998g in 250ml of water, adjust pH to 3.8]
2. the next day, dissolve 0.066g of lead nitrate in 10ml of aspartic acid and adjust the pH to 5.5 with 1N KOH, place it at 60°C for 30’)

- Wash in water (5x3’)

- Stain in lead aspartate for 30’ at 60°C

- Wash in water (5x3’)

- Dehydrate the sample on ice with cold ethanol graded series:
   20%, 50%, 70%, 90%, 100% 2x, 5’ each, and then with ice-cold acetone for 10’ at RT

- Infiltrate the sample with 25%, 50%, 75% Durcupan resin in acetone (2h each) at RT and with 100% Durcupan resin ON at 4°C

- The next day, infiltrate the sample with 100% Durcupan resin at RT (2x2h minimum)

- Process with the embedding with 100% Durcupan resin

- Cure the resin-embedded samples at 60°C for 24-48h

Figures
Click image for closeup view Click pictures for new window with figure and legend, click again for high resolution image

EMSTSFIG 1: Essential steps in embedding and trimming small pieces.
OTOFixRehydrationFIG 1: Low power view of hypodermis at midbody with dorsal side and muscle quadrant to the left and ventral to the right. Excretory canal and lateral nerve in the middle close to the pseudocoeolom. Stainingshows good contrast for many organelles.
EMSTSFIG 2:Thin sections floating behind diamond knife.
OTOFixRehydrationFIG 2: High power view of hypodermis. A large meisosome is at center left with a small multivesicular body found lower in the panel. Cytoplasm and organelles show a good range of contrast.

References

Aggad, D., Brouilly, N., Omi, S., Essmann, C.L., Dehapiot, B., Savage-Dunn, C., Richard, F., Cazevieille, C., Politi, K., Hall, D.H., Pujol, R., and Pujol, N. 2023. Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis. Elife 12: e74906. doi: 10.7554/eLife.75906

Deerinck, T.J., Bushong, E.A., Ellisman, M.H. and Thor, A. 2022. Preparation of biological tissues for serial block face scanning electron microscopy (SBEM) V.2. protocols.io 10.17504/protocols.io.36wgq7je5vk5/v2

Hall, D.H., Hartweig, E. and Nguyen, K.C.Q. 2012. Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107: 93-149. doi: 10.1016/B978-0-12-394620-1.00004-7

 
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