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squid-fix-stain.md

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Squid fixation and staining for visualization

After completing an aggregation or other squid colonization protocol, follow this procedure to fix squid and apply any tissue stains.

Reagents

2x mPBS - also called marine PBS

100mM sodium phosphate buffer, 0.9 M NaCl pH 7.4

  1. Make stock solutions
    • Stock solution A: 69 g monobasic sodium phosphate monohydrate (NaH2PO4 · H2O) in DI water to total volume of 1000 ml (0.5 M)
    • Stock solution B: 71 g dibasic sodium phosphate (Na2HPO4) in DI water to a total volume of 1000 ml (0.5 M)
  2. Combine 38 ml of A and 162 ml of B and check pH. Adjust to 7.4
  3. Add DI water up to 800 ml
  4. Add 52.6 g NaCl (0.9 M)
  5. Add DI water up to 1000 ml
  6. Store at 4C

To make 1X mPBS (50mM sodium phosphate buffer, 0.45 M NaCl, pH 7.4), mix 500ml of of 2X mPBS with 500ml DI water.

4% PFA fixative in 1X mPBS

Mix the following in a 50 ml conical

  • 10 ml 16% PFA (Electron Microscopy Sciences 16% PARAFORMALDEHYDE AQ SOLUTN (MeOH free), 10 x 10ml # 50-980-487)
  • 10 ml DI water
  • 20 ml 2X mPBS

Store at 4C

1X mPBS with 1% Triton-X

Mix the following in a 50 ml conical

  • 50 ml 1X mPBS
  • 500 ul Triton-X

Toto-3 stain solution

Mix the following in a 2ml microtube

  • 1 ml 1X mPBS with 1% Triton-X
  • 1.5 ul Toto-3

Use immediately and do not store. Be careful to limit exposure of stain to light.

WGA-633 1 mg/ml stock

WGA-633 (Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate) is Molecular Probes W21404
Mix 1 mg of powder into 1 ml DI water.
Store at -20 and protect from light.

Fixation of Squid

  1. Upon completion of squid experiment, anesthetize squid in 2% EtOH. This can be done in a 50 ml conical tube with 25 ml FSIO and 500 ul EtOH.
  2. Once squid are anesthetized, transfer to 4% PFA in 1x mPBS. This can be done in 3 ml conical tube with all squid per condition in the same tube. Wrap tube with foil and place on the rotator in the cold room for two nights.
  3. Wash squid with 1X mPBS 4 times for 15 minutes on 4C rotator.
  4. Dissect squid by opening mantel and removing funnel
  5. Proceed with imaging or further staining.

Staining of squid

Notes:

  • Multiple stains can be applied in parallel if the timing works out. Alternately, can wash out one stain and then apply the other stain.
  • Can likely adapt any of these stains to live or dead squid. Below are protocols that have been used reliably.

Counterstaining squid with Toto-3

This will stain the squid tissue red. Protocol is for fixed samples.

  1. Once squid is fixed and dissected, stain with Toto-3 (1:650 in 1X mPBS with 1% Triton-X). This is done in 1 ml of stain in a 2 ml microtube. All squid per condition may be placed in a single tube.
  2. Incubate for 1 hour in dark with motion (wrap tubes with foil and use Hula Mixer in microscope room).
  3. Rinse 2 times for 15 minutes with 1X mPBS with 1% Triton-X
  4. Rinse 2 times for 15 minutes with 1X mPBS
  5. Place squid on depression slide and image.

Counterstaining squid with CellTracker Orange CMRA

This will stain the squid tissue red (alternative to Toto-3). Protocol is for live samples.

  1. Add 1:1000 of 1 mM CellTracker Orange stock to Drosophila vial containing squid. Incubate 2-5 min for light organ surface or 30 min for crypts.
  2. Wash 3x in FSIO.
  3. Anesthetize squid in 2% EtOH and dissect for visualization.

Staining mucus with WGA-Alexa 633

This will stain mucus with far red. Protocol is for live samples.

  1. Add 1:100 of 1 mg/ml stock of WGA-Alexa 633 to Drosophila vial containing squid. Incubate 10 min for light organ surface or 30 min for crypts.
  2. Wash 3x in FSIO.
  3. Anesthetize squid in 2% EtOH and dissect for visualization.