Anti-Serotonin protocol with detailed notes

Last modified: 31 November 1999

1) Wash worms off plate with M9, put into 1.5 ml microcentrifuge tubes, spin down 30 sec in mini-centrifuge, remove supernatant.

• Note: One 60 mm plate with a dense, well-fed culture has plenty of worms for a staining experiment.

2) Rinse 2-3x with M9, spinning down and removing supernatant

• Note: One pasteur pipet-full is ~1.5 ml. Remove supernatant consistent with retaining as many worms as possible. In this protocol, "Rinse" always means adding the maximum fluid that will fit in the tube, i.e., one pipet-full.

3) Fix overnight (ON) at 4 degrees C in 500 ul of 4% paraformaldehyde/PBS.

• Note: Paraformaldehyde is somewhat toxic and probably carcinogenic. Work with it in the hood with gloves and lab coat.
• Note: Over-fixation kills staining. Staining seems to work better with 12-16 hr fixation vs. 24 hr. Two days of fixation abolishes staining.
• Note: 4% paraformaldehyde can be kept in the freezer for months. It can be thawed and used for about 2 weeks if kept at 4 degrees C. If a precipitate has formed If in a hurry, thaw tube of 4% paraformaldehyde in hot water; be sure to cool on ice before putting on worms. Hot fix kills all staining! (Also, if a precipitate has formed, vortex the solution vigorously. Heating the solution again to 60-70° C may be necessary if substantial precipitate remains.)

4) Rinse 3x in 0.5% Triton X-100/PBS.

• Note: Put the paraformaldehyde into the waste container in the hood.
• Note: After this rinse, the fixed worms can be kept at 4 degrees C for several days if necessary before proceeding, perhaps up to weeks. For preps with marginal staining, however, it is probably better not to wait.

5) Incubate ON at 37 degrees C in 500 ul of 5% beta-ME/1% TX-100/0.1 M Tris, pH 7.4 (Usually done rocking on Nutator in small incubator. Use "Sherlocks" or parafilm to seal tops).

• Note: beta-ME (beta-mercaptoethanol aka 2-ME) is extremely smelly and somewhat toxic. Work with it in the hood with gloves and lab coat. Container with the b-ME solution must be kept tightly closed or b-ME will evaporate. Make small batches in a glass container; if staining stops working it could be from loss of potency of this solution from evaporation.
• Note: Overtreatment at this step (e.g., 2 days or more) will also abolish staining.

6) Rinse 2x in 1% TX-100/0.1 M Tris, pH 7.4

• Note: Leave used pipets and tips in the hood for a day or two before discarding (the beta-ME will evaporate in that time).

7) Rinse 1x in collagenase buffer (1mM CaCl2/1% TX-100/0.1 M Tris, pH 7.4)

8) Incubate 30 min - 8 hr at 37 degrees C in 2000 Units/ml Collagenase type IV (SIGMA C5138) in collagenase buffer (Usually done rocking on Nutator in small incubator. Use "Sherlocks" or parafilm to seal tops).

• Note: Usual volume is ~200 ul, but this is usually adjusted depending on mass of collagenase aliquot used. Most batches of collagenase have ~500 U/mg, so typically you need ~4 mg/ml.
• Note: Digestion time is determined by examining 1-2 ul drops of worms (under the dissecting microscope) taken from the tube at regular intervals, starting as early as 15-30 min. When adult hermaphrodites begin to break open, stop treatment. Once a time is determined for a batch of collagenase, one usually needn't monitor digestion. Digestion time should be re-checked when staining different nematode species; this is sometimes also necessary for different C. elegans mutant strains.

9) Rinse 3x in 0.5% Triton X-100/PBS

10) Incubate for 1 hr at RT in 100 ul of 1% BSA/0.5% Triton X-100/PBS

• Note: This step is intended to block non-specific Ab binding.
• Note: BSA: Bovine serum albumen, Fraction V (Sigma A-9306), essentially immunoglobulin free for antibody solutions.

11) Incubate ON at RT in 1:100 anti-serotonin in 1% BSA/0.5% Triton X-100/PBS.

• Note: Although this incubation is typically performed ON at RT, this step can also be shortened to 1-2 hr at 37 degrees C.
• Note: Anti-serotonin is rabbit antiserum to serotonin (paraformaldehyde-conjugated to BSA; commercial antibodies are typically made to glutaraldehyde-conjugated serotonin) from Henry Steinbusch (H.Steinbusch@np.unimaas.nl), Maastrict University, Netherlands. USDA importation permit is no longer required for shipments, but appropriate endorsements must accompany shipment (see USDA Guidelines for Importation #1103).
• Note: Remove as much supernatant as possible (without losing worms) prior to adding the primary antibody. Add as little as 10 ul per tube of 1:100 Ab solution; although it is not practical to make less than 50 ul of solution (i.e., 0.5 ul stock antiserum to 50 ul diluent).
• Note: Anti-serotonin is frozen at -70 degrees C in 5 ul aliquots. Do not refreeze once thawed. It can safely be stored for many weeks at 4 degrees C. When finishing a tube, wash it out with the diluent to get as much Ab as possible. (It costs $180 for 50 ul!)

12) Rinse 2x in 0.5% Triton X-100/PBS

13) Incubate for 1 hr in 0.1% BSA/0.5% Triton X-100/PBS

14) Incubate 2-4 hr at 37 degrees C with 50 ul of 1:100 rhodamine-conjugated goat anti-rabbit IgG in 1% BSA/0.5% Triton X-100/PBS.

• Note: This incubation can also be performed ON at 4 degrees C.
• Note: Keep secondary antibody covered (e.g., with foil) at 4 degrees C for weeks. Aliquots are frozen at -70 degrees C for long-term storage. Do not refreeze once thawed.
• Note: Rhodamine-conjugated Abs fade much more slowly than those with FITC. Strictly speaking, we are actually using TRITC-conjugated antibodies (related to genuine rhodamine, but superior).

15) Rinse 3x in 0.5% Triton X-100/PBS

16) Rinse for 1 hr in 0.1% BSA/0.5% Triton X-100/PBS (You can typically skip this step and look right away, usually with a slightly higher background; if stopping here, do add 0.1% BSA solution.)

17) Mount on agarose pad/coverslip and view with epiflourescence

• Note: After the collagenase step, worms are often very fragile. Slightly sliding the coverslip after mounting on agarose pad can completely destroy the worms, leaving only shreds and pharynges.
• Note: Worms can typically be kept in tubes at 4 degrees C in the dark after staining and viewed for several days later without significant fading.

Other Notes:
In general, when rinsing, remove as much supernatant as possible without losing worms. Err on the side of removing less to retain worms. One exception is prior to adding the primary antibody, when you want to remove as much supernatant as possible to have the highest effective concentration of Ab.

You can add Sodium Azide (NaN3, final concentration 0.1 - 0.05%) to most or all solutions to keep things from growing (then they need not be autoclaved).

     [final]                    ( MW   )     10x        (for 1x)

     150 mM    NaCl             ( 58.44)     87.7 g    (8.77 g)

     8.4 mM    Na2HPO4.7H2O     (268.07)     22.5 g    (2.25 g)

     1.8 mM    NaH2PO4.H2O      (137.99)      2.5 g    (0.25 g)