Formaldehyde induced fluorescence (FIF) in C. elegans with notes

Last modified: 8 July 2014

FIF was first used in C. elegans to detect dopamine, published by Sulston et al., 1975. They got it working so reliably, apparently, that they were able to do a clonal mutant screen for FIF-deficient worms. Most others, perhaps not in the herculean mold of the legendary founders of the field, found the original protocol imposing and difficult to make reliable. I once received a photocopy of Bob Horvitz's hand-written, detailed protocol and started by transcribing the legible parts (most). The protocol is a bit intimidating, and involves a precisely-milled pair of aluminum blocks, vacuum dessication, alternating heating and cooling with a heat lamp, liquid nitrogen cooling, generating formaldehyde vapors under precise 70% humidity (generated with 33% concentrated H₂SO₄ - sulfuric acid) over several hours and an overnight step, etc. I've posted the Horvitz FIF protocol transcription just for fun & historical interest.

In Beth Sawin's 1996 dissertation (Horvitz lab) appears a much easier, reliable protocol for C. elegans that takes only minutes. Robyn Lints first published this protocol in the form seen below, and provided me some important pointers for making it work, which I share here along with some additional observations. So, thanks to Beth and Robyn, the rest of us can do FIF, too.

Formaldehyde-induced fluorescence

Whole animals were stained for dopamine by means of a formaldehyde-induced fluorescence (FIF) technique (Sulston et al., 1975; Jagdale and Gordon, 1994) as modified by Sawin (1996). One to five animals were placed on a microscope slide in a 5 µl drop of paraformaldehyde solution (4% paraformaldehyde in 0.1 M Na2HPO4/KH2PO4 buffer, pH 7.2) and incubated at room temperature for 5 minutes. Excess liquid was wicked off with a strip of 3 MM paper and the slide was heated for 10 minutes on an aluminium block at 96-100^oC, and then cooled to room temperature. A drop of glycerol and a coverslip were placed on top of the treated worms. Fluorescence was observed at X1000 with a Zeiss 487905 filter set (excitation, 395- 440 nm; emission, 470 nm long pass).

Summary version
1. 1-5 worms placed on slide in 5 µl PFA soln, 5 min RT
2. Wick away excess liquid (e.g., with 3MM paper)
3. Heat 10 min on block at 96-100^oC
4. Cool to RT
5. Add one drop glycerol, coverslip
6. View with appropriate fluorescence filter

FIF Protocol with Loer lab detailed notes:

• Note: Incubation time is maximum 5 min - begin step 2 so that all PFA is gone no later than 5 min; e.g. begin removing at 3.5 - 4 min). Over-fixation eliminates staining; err on the side of less than 5 min. Unlike in the Lints protocol, we have found one can actually put many, many worms in the drop via picking w/bacteria and swishing into the drop. Bacteria don't seem to interfere. Try to spread the worms around in the drop. For us, small larvae (L1/L2) work best; permability is likely an issue. To get larger/older C. elegans or less permeable species (such as Pristionchus) to work well/reliably, worms may need to be cut open in the PFA solution (not particularly easy). We have used this on several other free-living nematode species (Rivard et al., 2010). In some it works very well, showing lots of neurites as well as somas.

• Note:According to Robyn Lints, pH of the PFA solution is critical - so, as indicated, it should be made in a buffer solution. Long term, I keep PFA frozen at -20^oC - seems good indefinitely for antibody staining or FIF. After thawing and storing at 4^oC, for antibody staining (i.e., anti-serotonin) use within 2-4 weeks; for FIF it may be good much longer, perhaps months.

• Note: We make very long, pointy wedges of 3MM type paper and begin wicking at 3.5 - 4 min. With practice, one can carefully wick away liquid while leaving most worms. Having the drop well spread on the slide (just smaller than the coverslip size) makes it easier to wick away liquid while leaving worms behind.

• Note: We have a temperature block held constantly at 98^o C. On a typical small temp block, one can have up to 3 slides at a time heating; this works as one can only process about three at a time (progressively) with the 5 min PFA incubation/10 min heating step.

• Note: Briefly rest the slide on the bench or stereoscope stage - the slide cools very rapidly - in a few seconds. This happens in the time needed to get started on step 5, so there is no need to wait.

• Note: Use a trace of glycerol (perhaps 1-2 µl or less), just enough to 'wet' the prep under the coverslip (we use 18 mm or 22 mm square), with the glycerol slowly spreading under the coverslip by capillary action. With too much glycerol, the coverslip tends to slide around during viewing. Just dip a pasteur pipet or wooden applicator or pipet tip in the 100% glycerol. Then touch the slide just below the prep and lower the coverslip over the prep starting at this point.

• Note: The traditional filter set is Zeiss 487905 (excitation, 395- 440 nm; emission, 470 nm long pass), but this is apparently no longer available. We use the Chroma 11003v2 Blue/Violet filter set. [We also tested the Chroma 11005v2 Violet filter set. Signal seemed about the same, but the dopamine cells seemed a more distinctly different color with 11003v2, and photographed well with our camera.] Specs on the filter set can be found on the Chroma website.

  Background fluorescence is often very high, particularly from the gut. With the filter we use, cuticle and body fluorescence is yellow-green whereas dopamine cells are light blue / blue-green. As the gut fluorescence can also be more blue in color, cells within the body (such as PDE) can be more difficult to see clearly. Cells are much easier to see in the head and tail (which lack gut, of course). There is additional autofluorescence in the male tail, although it tends to be more yellow. Because the prep involves drying, the worms can be quite flattened, and so anatomical relationships may be distorted. Cells may appear in a single plane of focus (or pretty close to it). In good preps, some neurites may be seen and/or staining in the nerve ring. Using this protocol, we do NOT see green serotonin FIF, even in the NSMs (as it was first described by Horvitz et al.,1982).

  Not sure how long the fluorescence lasts, but my sense is that preps should be viewed immediately. May be good for a few hours, but probably not overnight.