OTO Fixation for SEM and Blockface Imaging
by Hall, Hartwieg and Nguyen
- Pick worms into a shallow type A HPF hat into a mixture of E. coli and 10% BSA. Fill the hat exactly, leaving no voids, then treat the top (blank) hat with hexidecene and close before freezing in the HPF device.
- The fast-frozen samples are placed into a shell vial containing 2% osmium tetroxide in acetone and placed into the freeze substitution device, set first at -90°C for 106 h, then slowly warmed to -30°C to hold for 16 h, then slowly warmed to 0°C. (See HPF methods section for more details.)
- Wash 4X with 100% acetone while at 0°C.
- Stain in 1% TCH in acetone at rt for 60 min (pre-filter this solution and keep in the dark).
- Wash 4X to 8X in acetone over 60 min at rt.
- Stain in 2% OsO4 in acetone for 60 min at rt.
- Wash 4X in acetone for 15 min at rt.
- Stain in 1% UAc in 90% acetone/ 10% methanol, 60 min in the dark at rt (filter before use).
- Wash 4X in acetone for 15 min at rt then transfer samples in Microporous Capsule, type C.
Dehydration and Infiltration
Place closed Microporous Capsule containing sample into a snap-cap vial containing methanol.
- Rinse 2X in 100% acetone.
- Start infiltration in 3:1 acetone:resin for 15 min at rt. Place snap-cap vial on rotator.
- Change to 2:1 acetone:resin for 3.5 h on rotator.
- Change to 1:1 acetone:resin overnight on rotator.
- Change to 1:3 acetone:resin 4 h on rotator.
- Change 4X in 100% resin over the course of 24 hrs on rotator.
- Embed between two pieces of ACLAR film and cure for one day at 60°C.
- Cut out desired sample from ACLAR and reposition in an embedding mold with fresh resin to embed the samples.
- Polymerize for 48 h at 60°C.
Resin Mixture: Hard Plus resin 812 kit (EMS catalogue # 14115).
TCH Working Solution:
10% TCH stock: 0.5 g TCH in 5 ml distilled water
Working solution: add 300 µl TCH stock into 15 ml methanol, then filter to get a 0.2% working solution in methanol.
This method is similar to what one currently does for the best TEM ultrastructure (see HPF method), except that extra steps have been added to add more heavy metal into the block for added contrast. This added contrast helps the SEM imaging, and may eliminate any need to post-stain the thin sections. The same protocol can be adapted for any SEM viewing technique, including ATUM thin sections, and FIB/SEM or 3View “Denkotome” blockface imaging. The outline of these steps follows the work of Deerinck and Ellisman as designed for mammalian tissues (Deerinck et al., 2010). We are grateful to JoAnn Buchanan, who first recommended the OTO method to us, and suggestions from Shegeki Watanabe for adapting the protocol for samples fixed by HPF, where all steps must be miscible in organic solvents, not aqueous.
Briefly: After a high pressure freeze fixation to capture the ultrastructure as perfectly as possible, the osmium stain is enhanced by the method of Hanker and colleagues (Seligman et al., 1966). Sodium thiocarbohydrazide (TCH) is used to bind the original osmium stain, after which a second round of osmium stain is applied: thus “OTO”. We have tried both OTO and OTOTO protocols to produce much more highly stained worms for SEM imaging. There are many possible variations to compare, including alternate first fixatives (aldehydes, tannic acid or UAc).
Deerinck, T.J., Bushong, E.A., Thor, A. and Ellisman, M.H. 2010. A new protocol for preparation of biological specimens for serial block face scanning electron microscopy. Microscop. Microanal. 16(sup2): 1138-9. Article
Hall, D.H., Hartweig, E. and Nguyen, K.C.Q. 2012. Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107: 93-149. Abstract
Seligman, A.M. Wasserkrug, H.L. and Hanker, J.S. 1966. A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxide-fixed tissue wit osmiophilic thiocarbohydrazide (TCH). J. Cell Biol. 30: 424-32. Article