Table 1.

Troubleshooting table

Step	Problem	Possible Reason(s)	Solution
Reagent Setup: resin mixture	Resin components cannot be stirred to form a homogeneous mixture	The viscosity of resin components, particularly Araldite 502, can pose problems for mixing by stirring.	Use half the amounts of all components listed, and dispense them into a 50 ml conical centrifuge tube. Cap tube, and shake vigorously. Don’t worry about bubble formation, but allow bubbles to dissipate before use. Warm individual resin components to 60°C, prior to mixing (this will reduce their viscosity)30.
1	Worms contract excessively or curl when submerged in fixative	Worms insufficiently chilled prior to fixation	Allow worms to cool a few minutes longer prior to fixation.
9	Specimens do not completely blacken during secondary fixation	Osmium tetroxide solution is old or weak.	Use a fresh vial of OsO4; fresh OsO4 should be pale yellow, not clear or (worse) brown-black. Check that OsO4 is not past its expiry date. Try OsO4 from a different lot number or supplier.
14	Acetone and resin do not mix uniformly, or mixture is cloudy.	Acetone is contaminated with water.	Store acetone over molecular sieves, and ensure that sieves have been regenerated as described in Reagent Setup.
21	Specimens cannot be oriented as desired in embedding molds	Even the most viscous epoxy resins are too fluid for specimen to remain in an unstable orientation, unsupported.	Use flat embedding molds, rather than capsules Pre-embed specimens in agarose after secondary fixation22, and trim agarose block so that it settles in desired orientation in the mold After the resin has cured, cut out a piece of the block containing the specimen, and glue it to another block in the desired orientation, using 5-minute epoxy glue.
23	Resin fails to cure, or remains soft.	Carryover of acetone from infiltration steps (20–22) One or more resin components have degraded. BDMA not used in sufficient quantity. Oven/incubator temperature too low. Insufficient time allowed for resin to cure.	Ensure that infiltration solutions are completely aspirated between steps. Use fresh resin components. BDMA is often most effective at concentrations of 3% (v/v) or more. Workers accustomed to working with other accelerators should not balk at adding so much BDMA. Test that oven/incubator reliably holds at a steady 60°C. Allow resin to cure at 60°C for at least 72 h.
Problems observed after completion of the protocol	Soft spots within specimen in block, or holes in sections.	Incomplete infiltration, due to:
		Water contamination in dehydrating solvents	Ethanol and acetone used in final dehydration (steps 11–13) should be stored over active molecular sieves.
		Incomplete dehydration of specimens (steps 11–13)	In step 12 add a third 30 min-incubation in fresh absolute ethanol.
		Insufficient time for infiltration with resin	Lengthen infiltration incubations in step 14 to 75 min each and/or use the overnight infiltration option for the 3:1 resin:acetone (step 14).
	Tissue in sections appears to be mechanically damaged.	Tearing or crushing when cutting a large worm into smaller pieces (step 5)	Ensure scalpel blade is fresh and sharp; wipe with tissue between cuts Use “guillotine” rocking motion to cut (Supplementary Figure 2a).
		Rough handling of brittle specimen after secondary fixation (steps 11 – end)	Avoid directly touching specimens after secondary fixation.
		Sections were cut from a location too close to the cut surface, in a worm piece (during microtomy, after completion of the protocol)	Cutting the worms in step 5 will inevitably damage tissue near the cut sites. Prior to cutting sections on the microtome, trim the block at least 50–100 μm beyond the cut surface, to ensure you are sectioning undamaged tissue.
	Poor fixation	Aged fixative components (Supplementary Figure 1)	Use fresh fixatives, or try a different supplier, or different lot from the same supplier.
		Homemade solutions (e.g. cacodylate buffer or formaldehyde) behave differently than commercially available solutions used by authors.	Test commercially available solutions for efficacy, or assay slight modifications of homemade solutions. See the note on buffer preparation and osmolarity under EM buffer in the Reagent Setup section.
		Suboptimal buffer conditions – pH or osmolarity.	Check that buffer is near pH 7 or slightly higher. Adjust buffer concentration. For example, if cells appear shrunken (Figure 2), try reducing the cacodylate concentration in the EM buffer from 70 to 60 mM.
		Specimens too large for fixatives to penetrate and act on tissue.	Cut specimens into smaller pieces. Alternatively, try extending the primary fixation time by 1h, and re-assess results. Check for penetration of OsO4 to centre of specimen by looking for blackening throughout specimen, and extend time for secondary fixation to 2h if fixative has not fully penetrated.
	Insufficient contrast of features when observed by TEM.	En bloc staining or post-staining of specimens not done, or insufficient.	Try staining specimens en bloc with uranyl acetate, e.g. add 2% (wt/vol) uranyl acetate to the 20% (vol/vol) ethanol dilution used for dehydration, and extend this step to 2–3h. Post-stain sections on grids with lead salts. We routinely use fresh lead citrate, as described by Venable and Coggeshall.51