Neuromelanin staining (Fontana-Masson staining)+ TH-DAB staining on midbrain organoids
Hariam Raji, michela.deleidi, Federico Bertoli
Abstract
Fontana-Masson staining is a silver staining technique that is commonly used to identify melanin-containing cells. We combined it with DAB-TH staining that works by using an antibody to detect the presence of TH, followed by a reaction with a substrate (DAB) that results in the formation of a brown-colored product at the site of the antigen-antibody interaction.
Attachments
Steps
Neuromelanin staining
Incubate human midbrain organoid sections in a fresh solution of 3:1 methanol (MeOH)/3% hydrogen peroxide at Room temperature for 0h 20m 0s.
Wash the slides and block it.
Wash the slides in PBS for 0h 5m 0s. (1/3)
Wash the slides in PBS for 0h 5m 0s. (2/3)
Wash the slides in PBS for 0h 5m 0s. (3/3)
Then, block with NGS 10% in PBS+ Triton-X 0.2% for 1h 0m 0s at Room temperature.
Apply primary antibodies in NGS 5% in PBS+ Triton-X 0.2% solution 1h 0m 0s at 4°C.
Next, wash slides.
Wash the slides in PBS for 0h 5m 0s. (1/3)
Wash the slides in PBS for 0h 5m 0s. (2/3)
Wash the slides in PBS for 0h 5m 0s. (3/3)
Apply secondary antibodies to NGS 5% in PBS+ Triton-X 0.2% solution for 1h 0m 0s at Room temperature.
Prepare ABC solution from Vectastain according to the manufacturer’s instructions (VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) PK-6100) and apply to sections for 1h 0m 0s at Room temperature.
Wash the slides.
Wash the slides in PBS for 0h 5m 0s. (1/3)
Wash the slides in PBS for 0h 5m 0s. (2/3)
Wash the slides in PBS for 0h 5m 0s. (3/3)
Prepare DAB solution according to the manufacturer’s instructions by diluting in 50mL of 1x PBS with 50µL of 3% H2O2.
Apply DAB solution to the sections at Room temperature for 30 seconds to 0h 12m 0s depending on when the visible reaction occurred.
For the visualization of neuromelanin, use the Fontana-Masson stain kit, according to the manufacturer’s instructions. (Fontana-Masson Stain Kit; Sigma‒Aldrich-HT200).
Eventually mount slides with synthetic resin.
