Jul 21, 2018

Public workspaceSudan Black staining of zebrafish larvae

PLOS Pathogens
DOI
dx.doi.org/10.17504/protocols.io.rced2te
  • 1University of Wisconsin - Madison
Emily Rosowski
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DOI: dx.doi.org/10.17504/protocols.io.rced2te
External link: https://doi.org/10.1371/journal.ppat.1007229
Protocol Citation: Emily Rosowski 2018. Sudan Black staining of zebrafish larvae. protocols.io https://dx.doi.org/10.17504/protocols.io.rced2te
Manuscript citation:
Rosowski EE, Raffa N, Knox BP, Golenberg N, Keller NP, Huttenlocher A (2018) Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing. PLoS Pathog 14(8): e1007229. doi: 10.1371/journal.ppat.1007229
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 27, 2018
Last Modified: July 21, 2018
Protocol Integer ID: 13414
Keywords: neutrophil staining
Abstract
Stains zebrafish larvae with Sudan Black B, which marks neutrophil granules.
Materials
MATERIALS
ReagentPhenol
ReagentEthanol 100%
ReagentSudan black BMerck MilliporeSigma (Sigma-Aldrich)Catalog #199664
ReagentTween20
Reagent16% Formaldehyde, methanol-freeFisher ScientificCatalog #NC1040701
ReagentPhosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813
ReagentPotassium hydroxide pellets
ReagentHydrogen Peroxide, 30%Fisher ScientificCatalog #H325-500
Before start
You will need a rocker/nutator/rotator at 4ºC and at RT.
Make both 1x and 10x stocks of PBS from powder packets, in water.
Make 70% Ethanol solution.
Make PBS-Tween = 1x PBS + 0.1% (v/v) Tween20.
Make 10% (w/v) Potassium hydroxide (KOH) in water.
Make Sudan Black B stock and working solution:
Stock: 0.18% (w/v) Sudan Black B in 70% Ethanol (store at RT, good for 1+ yr)
Working solution: 10 ml stock + 40 ml 70% Ethanol + 50 μl Phenol (store at RT, good for 6+ mo)
Anesthetize larvae
Anesthetize larvae
1. Anesthetize larvae with Tricaine.
2. Transfer to 1.5 ml microcentrifuge tube.
Fix larvae
Fix larvae
1. Make fresh 4% formaldehyde in PBS:
  • 1 ml 16% formaldehyde
  • 400 μl 10x PBS
  • 2.6 ml H2O
2. Pipet as much liquid off of larvae in tubes as possible.
3. Add 1 ml 4% formaldehyde per tube.
4. Cover in aluminum foil, rock at 4°C overnight.
Stain larvae
Stain larvae
1. Wash larvae in 1 ml 1x PBS, >5 min, rocking at 4°C.
2. Repeat for a total of 3 washes.
3. Pipet as much PBS as possible off of larvae, add 1 ml working solution of Sudan Black B (see Guidelines- Before you start).
4. Cover in aluminum foil, rock at RT for 1 hour.
Wash and Depigmentation
Wash and Depigmentation
1. Wash larvae in 1 ml 70% ethanol, 5 min, rocking at RT.
2. Repeat for a total of 3 washes.
3. Rehydrate larvae by washing in PBS-Tween, 5 min, rocking at RT.
4. Pipet as much PBS-Tween as possible off larvae, add 1 ml Depigmentation solution:
  • 400 μl 10% KOH
  • 133 μl 30% H2O2
  • 3.467 ml H2O
5. Rock at RT for 5 minutes.
6. Take out a few larvae from tube, check depigmentation under a zoomscope. Pigment should be mostly lost, and sudan black signal should remain.
7. Wash larvae in 1 ml PBS-Tween, 5 min, rocking at RT.
8. Repeat for a total of 2 washes.
9. Store at 4ºC. For longer-term storage change liquid to 1x PBS (no Tween).