Re: Luxol Fast Blue-Holmes stain

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From:rzumbo <>
To:Curtis King <>,
Content-Type:text/plain; charset=US-ASCII

This is our method we use for combination Holmes/LFB

- Dewax & bring to water
- Wash in deionised water for 10min
- Place in strong silver solution for 3hours at room temperature in 
the dark
- Rinse in deionised water for 10 minutes
- Place slides in impregnating solution in covered coplin jar, leave 
overnight at 37degrees
- Remove slides from impregnating solution drain and place in 
reducing solution for 6 minutes
- Wash in tap water for 3 minutes
- Rinse in deionised water
- Tone in 0.2%gold chloride for 5 minutes
- Rinse in deionised water
- Place in 5% Oxalic acid until axons are black ( up to 45 minutes)
- Rinse in Deionised water
- Fix in 5% Sodium thiosulphate for 10 minutes
- Wash in tap water for 10 minutes
-Place sections in preheated Luxol Fast blue stain in a 45 degree 
oven for minimum of 4 hours
- Remove from oven allow to stand om bench for 15 minutes
- Rinse in 95% alcohol
- Rinse in deionised water
- Place in 0.05% Lithium carbonate solution for a few seconds
- Differentiate in 70% alcohol for 20-30 seconds
- Rinse in deionised water (check microscopically)
- Repeat lithium carbonate- 70% - water steps until myelin stands 
out on a clear background.
- Wash in water
- Dehydrate, clear, mount

1 Ampoule of Merck 0.1N Silver nitrate, dilute to 100mL

0.2M (1.24%) Boric Acid - 22mL
0.05 ( 1.9%) Borax (Sodium tetraborate) - 18mL
Mix in measuring cylinder and make up to 194mL
Add by pipette 4mL of 10% Pyridine and 2mL of 1mL silver nitrate

Sodium sulphite - 20g
Hydroquinone - 2g
Deionised water - 200mL
Mix, last 1 week

Luxol fast blue - 1g
95% alcohol - 1000mL
10% Acetic acid - 5mL

Rosalba Zumbo
Technical Officer ( Scientific)
Institute of Forensic Medicine
Sydney, Australia

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