Parasite cryo-embedding protocol.

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Image paths corrected during migration. All 4 images were linked as ../img/… in the source, one directory level too high, so none displayed. The source also contains a stray [img2] placeholder in step 4 of the measurement section, left over from editing.

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Materials

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Telling head from tail: under dissection microscopy, the tail will be slightly curved while the head will be straighter and rounder.

Clean all cryomolds, tools, and working surfaces with Eliminase prior to embedding.

Embed twice the number of specimens you need — this approach is prone to error given the number of steps and the precision required from sectioning through RNA extraction.

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Orienting and imaging nematodes for downstream spatial sectioning

Parasite staining

  1. In a 10 cm Petri dish, make a 1:5 dilution of 0.05% methylene blue in RPMI and mix with a pipette tip. 100 μL of 0.05% methylene blue in 400 μL RPMI is typical for Brugia adults and L3/L4 from all species.
  2. Prepare 3 or 4 pools of 100 μL RPMI in the Petri dish. These will be used to serially wash the L3/L4 or adults after staining, to remove excess methylene blue.
  3. For L3 and L4: take a group of L3 in a volume of 20 μL using a p20 pipette from a pool of washed L3, and drop them into the pool of diluted methylene blue. For adults: using a worm hook, take one adult at a time from a culture plate containing sorted and washed adults, and transfer to the pool of diluted methylene blue.
  4. Allow parasites to soak for two to five minutes. Monitor the colour change using the scope. You may need to add time if they are not stained completely.
  5. While the parasites are staining, prepare the dry ice for embedding: