Dec 18, 2023
Version 1
Cartilage staining V.1
- Satheeswaran Balasubramanian1,
- Ekambaram Perumal1
- 1Bharathiar University
- Fish behavior and physiology
- Zebrafish Research
Protocol Citation: Satheeswaran Balasubramanian, Ekambaram Perumal 2023. Cartilage staining. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3je5lk5/v1Version created by Satheeswaran Balasubramanian
Manuscript citation:
Balasubramanian S, Rangasamy S, Vivekanandam R, Perumal E. Acute exposure to tenorite nanoparticles induces phenotypic and behavior alterations in zebrafish larvae. Chemosphere. 2023 ;339:139681. https://doi.org/10.1016/j.chemosphere.2023.139681
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
We use this protocol and it's working
Created: November 25, 2023
Last Modified: December 18, 2023
Protocol Integer ID: 91415
Keywords: Cartilage, zebrafish, alcian blue
Funders Acknowledgements:
ICMR-SRF
Grant ID: No.45/51/2020-Nan/BMS
Abstract
The Alcian blue staining technique is widely used among developmental biologists to observe the development of cartilage and bone in zebrafish embryos and larvae. Alcian blue is a positively charged dye that stains the cartilage through an electrostatic interaction with negatively charged acidic mucopolysaccharides in the presence of magnesium ions (Scott et al. 1996).
Guidelines
This protocol is optimized for 5 days post-fertilization (dpf) of zebrafish larvae. It can be adapted for higher dpf.
Materials
| A | B | C | |
| Abbreviation | Chemical Name | Molecular Weight (g/mol) | |
| Na2HPO4 | Disodium hydrogen phosphate | 141.96 | |
| MgCl2 | Magnesium chloride | 95.21 | |
| KCl | Potassium chloride | 74.55 | |
| KH2PO4 | Potassium dihydrogen phosphate | 136.09 | |
| KOH | Potassium hydroxide | 56.11 | |
| NaCl | Sodium chloride | 58.44 | |
| dH2O | Distilled water | - | |
| EtOH | Ethanol | 46.07 | |
| PFA | Paraformaldehyde | 30.03 | |
| PBS | Phosphate-buffered saline | - | |
| PBST | PBS plus 0.1% Tween 20 | - | |
| MeOH | Methanol | 32.04 |
- 10X PBS stock; pH 7.4: To prepare 1 L stock, add 1.37 M (80 g) NaCl, 27 mM (0.2 g) KCl, 100 mM (14.4 g) Na2HPO4, and 18 mM (0.24 g) KH2PO4 in 800 ml of dH2O. Make up the volume to 1000 ml and autoclave. Store at room temperature. To prepare 1X PBS, add 100 ml of 10X PBS to 900 ml dH2O.
- 1X PBST: Add 0.1 ml of Tween 20 to 100 ml of 1X PBS and mix.
- 4 % PFA; pH 7.4: Dissolve 4 g of PFA in 80 ml of PBS at 60 °C, until the solution clears. Cool the solution and make it to 100 ml. To avoid repeated thawing and freezing, PFA can be aliquoted in 5 ml tubes and stored at −20 °C.
- Bleach solution: For 1 ml, add 100 μl of 30% solution of H2O2, 250 μl of 2% KOH and 650 μl of dH2O.
- Staining solution: For 1 ml, add 100 μl of 10X alcian blue (0.01%), 200 μl of 1 M MgCl2 (60 mM), and 700 μl of 100% EtOH (70%). a) 10X of alcian blue solution (8 GX; 05500, Sigma Aldrich): For 10 ml solution, add 10 mg alcian blue and 10 ml of methanol. b) 300 mM of MgCl2: Add 0.29 g of MgCl2 to 10 ml of dH2O. c) 100% EtOH.
- Storage solution: Keep 100% glycerol and 1% KOH (100 mg in 10 ml) separately.
a. 25% solution: For 1 ml, mix 250 μL glycerol, 250 μl of KOH, and 500 μl of dH2O.
b. 50% solution: For 1 ml, mix 500 μL glycerol, 250 μl of KOH, and 250 μl of dH2O.
Safety warnings
Make sure to read all Safety Data Sheets for the reagents. Hydrogen peroxide and paraformaldehyde solution must be prepared under a fume hood at all times.
Before start
- Every protocol needs to be validated in the laboratory when first introduced. The present protocol describes validation steps that were taken in the Molecular Toxicology lab, at Bharathiar University.
- Always wear personal protective equipment.
- All the steps are optimized in a 24-well plate for multiple groups such as control and treated.
Larval fixation
Larval fixation
3h
3h
At the desired stage, take the zebrafish larvae and wash in PBS for 5 minutes.
5m
Euthanize the larvae using the cold shock method (Keep at 4oC for 5-10 minutes).
10m
Transfer the euthanized larvae into 4% PFA and keep it in the rocker for 2 hours at room temperature (if required, it can be kept overnight at 4oC).
2h
Wash the fixed larvae with PBST for 5 minutes, two times.
10m
Dehydrate the larvae using 50% ethanol and keep it in the rocker for 10 minutes at room temperature.
10m
Staining
Staining
8h
8h
Add 1 ml of alcian blue staining solution to the dehydrated larvae and keep it in a rocker overnight at room temperature.
8h
Bleaching
Bleaching
40m
40m
Rinse the stained larvae once using PBST.
1m
Transfer the larvae to a bleach solution and incubate in dark at room temperature. Assess the bleaching process (using a microscope) and stop the reaction when the larvae become transparent (approximately 20-30 min for 4 dpf and above).
30m
Clearing
Clearing
9h
9h
Remove the bleach solution and rinse once using PBST.
1m
Clear the beached larvae with 25% glycerol and 0.25% KOH. Keep them in a rocker at room temperature for 30 minutes to overnight.
30m
Replace the solution with 50% glycerol and 0.25% KOH. Keep them in a rocker at room temperature for 2 hours to overnight.
8h
Storage
Storage
Store larvae in a solution of 50% glycerol and 0.1% KOH at 4oC. Avoid long-term storage as it will diminish the stain. Capture images of the cartilage using any bright field microscope (preferably a stereo microscope).
Results
Results
Expected result
Figure 1. Representative staining images of 5 dpf larvae. a) Whole larvae; b) head region (taken in molecular toxicology lab, Bharathiar University); c) Schematic representation of cartilaginous structures in the head region of zebrafish (adapted from Raterman et al. 2020).
Protocol references
Dr Mb Walker & Cb Kimmel (2007) A two-color acid-free cartilage and bone stain for zebrafish larvae, Biotechnic & Histochemistry, 82:1, 23-28, DOI: 10.1080/10520290701333558