Feb 15, 2022
Gallyas-silver stain
- Christiana.bjorkli 1,
- María osé Lagartos Donate2
- 1The Norwegian University of Science and Technology;
- 2University of Oslo
- Sandvig lab
Protocol Citation: Christiana.bjorkli , María osé Lagartos Donate 2022. Gallyas-silver stain. protocols.io https://dx.doi.org/10.17504/protocols.io.b44cqysw
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: February 15, 2022
Last Modified: February 15, 2022
Protocol Integer ID: 58212
Keywords: pathological staining in rodent, silver staining protocol, pathological staining, silver stain, gallya, staining protocol, rodent, human tissue
Abstract
Simplified Gallyas-silver staining protocol that works for pathological staining in rodent and human tissue
Solutions that can be made in advance and stored in a fume hood
Make 4% of NaOH (sodium hydroxide) - use gloves, store in fume hood
1g NaOH + 25ml H20
Make 2 x 600ml dH20 – use gloves, store in fume hood
1.5g ammonium nitrate + 1.2g silver nitrate + 3.6g of 4%NaOH
Safety information
Ammonium nitrate can cause an explosion with metals
Reaction between silver nitrate and ethanol is explosive
Reaction between silver nitrate and NaOH can form inflammable gases/vapors
Make acetic acid – use gloves, store in fume hood
0.5%: 3ml acetic acid in 597ml H20
0.1%: 0.6ml acetic acid in 600ml H20
0.05%: 50ml 0.5% acetic acid + 450ml H20
Make 0.2% potassium ferricyanide – can be stored up to a week, use gloves, store in fume hood
0.6g potassium ferricyanide in 300ml H20
Make 0.5% sodium thiosulfate - use gloves, store in fume hood
1.5g sodium thiosulfate in 300ml H20
Make 25%, 50%, 70%, 95% and 100% ethanol
Solutions that need to be prepared on the same day of staining
Make pyridine + acetic acid - use gloves, store in fume hood
100ml pyridine + 50ml acetic acid
Safety information
Pyridine is incompatible with rubber, plastics, and metals! Very important to use butyl-rubber gloves with this chemical
Make silver nitrate (add in this order) - use gloves, store in fume hood
100ml dH20
0.192g ammonium nitrate
0.2g silver nitrate
0.6ml of 4% NaOH
pH must be 7.5
Safety information
Ammonium nitrate can cause an explosion with metals
Reaction between silver nitrate and ethanol is explosive
Reaction between silver nitrate and NaOH can form inflammable gases/vapors
Make A + B + C (1L) - use gloves, store in fume hood
Solution A
5g anhydrous sodium carbonate + 100ml dH20
Solution B (add in this order)
100ml dH20 + 0.19g ammonium nitrate + 0.2g silver nitrate + 1g silicongulistic (tungolistic) acid
Solution C (add in this order)
100mldH20 + 0.19g ammonium nitrate + 0.2g silver nitrate + 1g silicongulistic (tungolistic) acid + 0.66ml 37% formaldehyde
A + B + C - use gloves, store in fume hood
50ml A + 37.5ml B + 37.5ml C (25±2°C)
Safety information
Ammonium nitrate can cause an explosion with metals
Reaction between silver nitrate and ethanol is explosive
Reaction between silver nitrate and NaOH can form inflammable gases/vapors
Formaldehyde may cause cancer
Consumables for staining
17 x glass petri dishes
20 x glass pipettes
1 x rubber pump for glass pipettes
Butyl rubber gloves
Lab coat with long arms
Protective eyewear
Face mask
Note
Need to use glass petri dishes and glass pipettes for each wash
Gallyas-silver staining
| Step | Reagent | Minutes | Comments | |
| 1 | dH20 | 3 or more | Put in dH20 while preparing other solutions | |
| 2 | Pyridine + acetic acid | 60 | On shaker | |
| 3 | 50% ethanol | 3 | ||
| 4 | 25% ethanol | 3 | ||
| 5 | 0.05% acetic acid | 2.5 | ||
| 6 | 0.1% acetic acid | 2.5 | ||
| 7 | 0.05% acetic acid | 10 | Leave longer if necessary, to prepare silver nitrate | |
| 8 | Silver nitrate | 60 | On shaker – make A+ B + C | |
| 9 | 0.5% acetic acid | 10 | ||
| 10 | A + B + C | 6 (15min human) | Do not let the tissue become dark, time is dependent on sample | |
| 11 | 0.5% acetic acid | 1 | New | |
| 12 | 0.2% potassium ferricyanide | 5 | On shaker | |
| 13 | dH20 | 1 | ||
| 14 | 0.5% acetic acid | 1 | New, can be reused | |
| 15 | 0.5% sodium thiosulfate | 2 | On shaker | |
| 16 | dH20 | 4 | ||
| 17 | dH20 | 4 | ||
| 18 | 0.5% acetic acid | 1 | Use the one from the previous step | |
| 19 | A + B + C | 3-4 | On shaker | |
| 20 | 0.5% acetic acid | Use the one from the previous step | ||
| 21 | 0.2% potassium ferricyanide | 3 | On shaker | |
| 22 | dH20 | 1 | ||
| 23 | 0.5% sodium thiosulfate | 2 | On shaker | |
| 24 | dH20 | 4 | ||
| 25 | dH20 | 4 | ||
| 26 | 0.5% acetic acid | 1 | New, can be reused | |
| 27 | A + B + C | 3-4 | On shaker | |
| 28 | 0.5% acetic acid | 1 | Use the one from the previous step | |
| 29 | 0.2% potassium ferricyanide | 10 | On shaker | |
| 30 | dH20 | 1 | ||
| 31 | 0.5% sodium thiosulfate | 2 | On shaker | |
| 32 | dH20 | Fast wash | ||
| 33 | dH20 | 5 | ||
| 34 | Dehydrate | 3 in each | 50% - 70% -95% - 100% - 100% ethanol | |
| 35 | Xylene x 2 | 3 in each | ||
| 36 | Coverslip |