Apr 05, 2024
Gallyas Silver Staining
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. Gallyas Silver Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo366zv4o/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 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94364
Keywords: ASAPCRN
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Abstract
Gallyas Silver staining in mouse brain sections stains degenerating neurites black.
Day 1
Day 1
For silver staining use the FD NeuroSilverTM Kit II (FD NeuroTechnologies INC Cat# PK301)
Staining of free-floating brain sections is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64)
The sections can be transferred between wells using a paint brush.
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
Incubate sections in 0.1M PB + 4% paraformaldehyde pH 7.4 for 7 days at 4°C
Cover dish in parafilm
Lightly shake sections during this step - we use a shaker in the cold room
Day 8
Day 8
Perform all silver staining steps in a chemical fume hood.
Take shaker into fume hood for this purpose.
Collect all silver stain waste in large beaker for hazardous waste disposal
Wash sections in miliQ H2O 2 times for 5 minutes each wash at room temp
While sections are washing make up the A/B solution
Make 80ml per dish : 40ml sol A + 40ml sol B
Wash sections in A/B solution for 2 times for 10 min each wash at room temp
Use 25mL solution for each wash
Wash sections in A/B solution + E for 10 min at room temp
8mL sol A/B + 1 drop solution E
For 1 dish: 24ml sol A/B + 3 drops sol E
Wash sections in C/F solution for 2 min at room temp
50mL sol C + 2 drops sol F
Use 25mL per wash
Wash sections in C/F solution for 4 min at room temp
Incubation longer than 4 min at this step decreases background and decreases signal. Shorter incubation increases signal but also increases background.
Wash sections in D/F solution 1 time for 5 min at room temp
25mL sol D + 1 drop sol F
Make 25mL per dish
Wash sections in miliQ H2O 2 times for 3 min each wash at room temp
Wash sections in 1X solution G 2 times for 5 min each wash
Dilute 10X sol G 1:10 in miliQ H2O
Make 80mL per dish
Store sections in 1X sol G
Use a petri dish filled with 1X sol G to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15)
This is easiest to do using a medium sized paint brush
Let sections dry at room temp after mounting
Protect slides from light
Clear sections in fresh xylene 3 times for 3 min each wash at room temp
DO NOT dehydrate in EtOH – it will cause loss of staining.
Use fresh xylene. Old xylene can contains residual EtOH from previous experiments. Residual EtOH will remove silver staining.
Use Entellan mounting medium (Electron Microscopy Sciences 14802) to coverslip slides
Apply one line of Entellan across the middle of the slide
Cover with glass coverslip and gently press down with forceps
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