TITLE: Gallyas Silver Staining
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
Gallyas Silver staining in mouse brain sections stains degenerating neurites black. 

[STEPS]
SECTION: Day 1
1. For silver staining use the FD NeuroSilverTM Kit II (FD NeuroTechnologies INC Cat# PK301)
SECTION: Day 1
2. Staining of free-floating brain sections is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64) 
SECTION: Day 1
2.1. The sections can be transferred between wells using a paint brush. 
SECTION: Day 1
3. Wash sections 35µm mouse brain sections in 0.1M Phosphate buffer (PB) 3 times (5 minutes each wash) at room temp to remove cryoprotectant solution
SECTION: Day 1
4. Incubate sections in 0.1M PB + 4% paraformaldehyde pH 7.4 for 7 days at 4°C
SECTION: Day 1
4.2. Lightly shake sections during this step - we use a shaker in the cold room
SECTION: Day 1
4.1. Cover dish in parafilm
SECTION: Day 8
5. Perform all silver staining steps in a chemical fume hood. 
SECTION: Day 8
5.1. Take shaker into fume hood for this purpose. 
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5.2. Collect all silver stain waste in large beaker for hazardous waste disposal
SECTION: Day 8
6. Wash sections in miliQ H2O 2 times for 5 minutes each wash at room temp
SECTION: Day 8
6.1. While sections are washing make up the A/B solution
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6.2. Make 80ml per dish : 40ml sol A + 40ml sol B
SECTION: Day 8
7. Wash sections in A/B solution for 2 times for 10 min each wash at room temp
SECTION: Day 8
7.1. Use 25mL solution for each wash
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8. Wash sections in A/B solution + E for 10 min at room temp
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8.1. 8mL sol A/B + 1 drop solution E
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8.2. For 1 dish: 24ml sol A/B + 3 drops sol E
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8.3. Wash sections in C/F solution for 2 min at room temp
SECTION: Day 8
8.4. 50mL sol C + 2 drops sol F
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8.5. Use 25mL per wash
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9. Wash sections in C/F solution for 4 min at room temp
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9.1. Incubation longer than 4 min at this step decreases background and decreases signal. Shorter incubation increases signal but also increases background.
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10. Wash sections in D/F solution 1 time for 5 min at room temp
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10.1. 25mL sol D + 1 drop sol F
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10.2. Make 25mL per dish
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11. Wash sections in miliQ H2O 2 times for 3 min each wash at room temp
SECTION: Day 8
12. Wash sections in 1X solution G 2 times for 5 min each wash
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12.1. Dilute 10X sol G 1:10 in miliQ H2O
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12.2. Make 80mL per dish
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12.3. Store sections in 1X sol G
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13. Use a petri dish filled with 1X sol G to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15)
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14. This is easiest to do using a medium sized paint brush
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15. Let sections dry at room temp after mounting
SECTION: Day 8
16. Protect slides from light
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17. Clear sections in fresh xylene 3 times for 3 min each wash at room temp
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17.1. DO NOT dehydrate in EtOH – it will cause loss of staining. 
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17.2. Use fresh xylene. Old xylene can contains residual EtOH from previous experiments. Residual EtOH will remove silver staining. 
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18. Use Entellan mounting medium (Electron Microscopy Sciences 14802) to coverslip slides 
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18.1. Apply one line of Entellan across the middle of the slide
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18.2. Cover with glass coverslip and gently press down with forceps
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18.3. Let slides dry in the fume hood for one hour – then dry on the bench overnight at room temp (protect slides from light) Gently remove excess Entellen with a razor blade