Anti-Serotonin protocol with detailed notes


Short version
Solutions for Anti-Serotonin staining
References

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.

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

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

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

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).

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

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).

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

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

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.

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

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).


Solutions for Anti-serotonin protocol

1M Tris, pH 7.4
10% Triton X-100 - This is much easier to work with than 100% Triton, which takes a long time to mix with water.
  1. Make up this stock by adding 10 ml Triton X-100 to 90 ml water.
  2. Put stir bar in bottle with water, then add Triton very slowly while stirring.
  3. Wash out pipet with solution at the end because much viscous Triton will be stuck to the inside of the pipet.

10X PBS (Phosphate Buffered Saline - Kenyon lab recipe)
     [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)

  1. dissolve in ~800 ml ddH2O
  2. adjust pH to 7.4 with approx. 13 ml 1N NaOH
    (=13 mMoles/1liter 10x buffer or 1.3 mMoles//1liter 1x buffer)
  3. adjust to 1 liter
  4. aliquot and autoclave

    Na2HPO4.7H2O = Sodium Phosphate, dibasic, heptahydrate (or if using Na2HPO4, use 11.94 g)
    NaH2PO4.H2O = Sodium Phosphate, monobasic

4% paraformaldehyde in PBS
  1. Heat ~80 ml dd water to 55-65 degrees C in beaker stirring in hood (use dedicated beaker, stir bar and 'hood thermometer').
  2. Add 4 g paraformaldehyde powder or flakes, stir until dissolved. Add a few drops of 1M NaOH if necessary to help the paraformaldehyde to go into solution.
  3. Solution should become clear. Don't boil or heat longer than necessary.
  4. Add 10 ml 10x PBS and make up to 100 ml.
  5. Aliquot 10 ml each in 15 ml centrifuge tubes and store in -20 degrees C freezer.

References for Anti-serotonin protocol

Neurotransmitter antisera were "invented" by H.W.M. Steinbusch et al. (1988). This protocol for C. elegans is derived from that originally published by Desai et al. (1988) with minor modifications (Loer & Kenyon, 1993). I have continued to modify the above protocol, and provide notes on variations that you can or cannot get away with.

Desai C, Garriga G, McIntire SL, Horvitz HR (1988) A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 336:638-46.

Loer CM, Kenyon CJ (1993) Serotonin-deficient Mutants and Male Mating Behavior in the Nematode Caenorhabditis elegans. J Neurosci 13: 5407-5417.

Steinbusch HWM, Wouterlood FG, deVente J, Bol JGJM, Berkenbosch F (1988) Immunohistochemical localization of monoamines and cyclic nucleotides. Acta Histochem S35:85-106.


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