Jan 31, 2024
Fontana-Masson staining
- Cristian González-Cabrera1,
- Csilla Novák1,
- Andrés auricio Jaramillo Flautero1,
- Celine Winter1,
- Matthias Prigge1
- 1Neuromodulatory Network Group, Leibniz Institute for Neurobiology, Magdeburg
- TeamPrigge
Protocol Citation: Cristian González-Cabrera, Csilla Novák, Andrés auricio Jaramillo Flautero, Celine Winter, Matthias Prigge 2024. Fontana-Masson staining. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmxoxl5b/v1
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: January 31, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94459
Keywords: Neuromelanin, Staining, pigments, ASAPCRN, masson staining protocol, other pigments in tissue sample, pigment analysis in tissue, pigment analysis, staining, other pigment, tissue sample, melanin, silver nitrate working solution, fontana, thorough washing steps between each incubation
Funders Acknowledgements:
ASAP
Grant ID: 020505
Abstract
The Fontana-Masson Staining Protocol is a detailed method for staining free-floating sections (20-50 microns) to detect melanin and other pigments in tissue samples. The procedure involves preparing a silver nitrate working solution, treating sections with triton, and sequential incubation in pre-heated silver nitrate solution, gold chloride, and thiosulfate solutions, with thorough washing steps between each incubation. The process concludes with sections being placed in PBS for mounting and counterstaining. This protocol is critical for histological studies in biology, particularly for pigment analysis in tissues.
Materials
Silver nitrate solution
Ammonium hydroxyde (concentrated)
PBS with 0.3% triton
Gold chloride solution
Thiosulfate solution
Distilled water
Tap water
Troubleshooting
Fontana-Masson staining
3h 14m 30s
Use free floating sections between 20-50 microns.
Silver Nitrate working solution:
- Prepare a 1:3 solution of silver nitrate solution in distilled water. Prepare enough solution for the amount of sections you want to stain.
- Add drop by drop concentrated ammonium hydroxide, while shaking, until the solution gradually turns transparent. Be careful and stop the addition of ammoniacal solution as soon as you get transparency.
10m
Place the sections to stain (up to 7-8 sections) in a 2 mL Eppendorf tube. Incubate the sections in 0.3% triton for two hours at room temperature while shaking
2h
Wash 3 times with distilled water for 5 min each.
15m
Pre-heat the silver nitrate working solution for 5 minutes at 60 °C .
5m
Incubate the sections in the preheated solution for 20 minutes at 60 °C .
20m
Wash the sections 3 times with abundant distilled water.
From now on, perform the following steps in individual sections:
Incubate the section with gold chloride solution. Apply enough solution to cover the section (approximately 100-150 µL ). Manually shake the tube for 30 seconds.
30s
Wash the section 3 times with abundant distilled water.
5m
Incubate the section with thiosulfate solution. Apply enough solution to cover the section (approximately 100-150 µL ). Manually shake the tube for 2 minutes.
2m
Wash with tap water for 2 minutes.
2m
Wash 3 times with distilled water for 5 min each.
15m
Place the sections in PBS for mounting, counterstaining, etc.
Results
Slices should now show an amplified pigmentation as show in figures