TITLE: Gallyas-silver stain
AUTHORS: Christiana.bjorkli  , María José Lagartos Donate

[DESCRIPTION]
Simplified Gallyas-silver staining protocol that works for pathological staining in rodent and human tissue 

[STEPS]
SECTION: Solutions that can be made in advance and stored in a fume hood
2. 
SECTION: Solutions that can be made in advance and stored in a fume hood
1. Make 4% of NaOH (sodium hydroxide) - use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
3. 
SECTION: Solutions that can be made in advance and stored in a fume hood
1. Make 4% of NaOH (sodium hydroxide) – sodium hydroxide – this is in the Kentros lab make in fume hood, use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
1.1. 1g NaOH + 25ml H20
SECTION: Solutions that can be made in advance and stored in a fume hood
2. Make 2 x 600ml dH20 – use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
2.1. 1.5g ammonium nitrate + 1.2g silver nitrate + 3.6g of 4%NaOH
 

SECTION: Solutions that can be made in advance and stored in a fume hood
3. Make acetic acid – use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
3.1. 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
SECTION: Solutions that can be made in advance and stored in a fume hood
4. Make 0.2% potassium ferricyanide – can be stored up to a week, use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
4.1. 0.6g potassium ferricyanide in 300ml H20
SECTION: Solutions that can be made in advance and stored in a fume hood
5. Make 0.5% sodium thiosulfate - use gloves, store in fume hood
SECTION: Solutions that can be made in advance and stored in a fume hood
5.1. 1.5g sodium thiosulfate in 300ml H20
SECTION: Solutions that can be made in advance and stored in a fume hood
6. Make 25%, 50%, 70%, 95% and 100% ethanol 
SECTION: Solutions that need to be prepared on the same day of staining
7. Make pyridine + acetic acid - use gloves, store in fume hood
SECTION: Solutions that need to be prepared on the same day of staining
7.1. 100ml pyridine + 50ml acetic acid
 

SECTION: Solutions that need to be prepared on the same day of staining
9. Make A + B + C (1L) - use gloves, store in fume hood
SECTION: Solutions that need to be prepared on the same day of staining
8. Make silver nitrate (add in this order) - use gloves, store in fume hood
SECTION: Solutions that need to be prepared on the same day of staining
8.1. 100ml dH20
0.192g ammonium nitrate
0.2g silver nitrate
0.6ml of 4% NaOH
pH must be 7.5 
 

SECTION: Solutions that need to be prepared on the same day of staining
9.1. 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)
 

SECTION: Consumables for staining
10. 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
 

SECTION: Gallyas-silver staining
11.  
StepReagent Minutes Comments 1dH203 or morePut in dH20 while preparing other solutions2Pyridine + acetic acid60On shaker350% ethanol 3 425% ethanol  3 50.05% acetic acid2.5 60.1% acetic acid 2.5 70.05% acetic acid 10Leave longer if necessary, to prepare silver nitrate8Silver nitrate60On shaker – make A+ B + C90.5% acetic acid 10 10A + B + C6 (15min human)Do not let the tissue become dark, time is dependent on sample110.5% acetic acid1New120.2% potassium ferricyanide5On shaker 13dH201 140.5% acetic acid1New, can be reused 150.5% sodium thiosulfate2On shaker 16dH204 17dH204 180.5% acetic acid1Use the one from the previous step19A + B + C3-4On shaker200.5% acetic acid Use the one from the previous step210.2% potassium ferricyanide3On shaker 22dH201 230.5% sodium thiosulfate2On shaker24dH204 25dH204 260.5% acetic acid1New, can be reused27A + B + C3-4On shaker280.5% acetic acid1Use the one from the previous step290.2% potassium ferricyanide10On shaker30dH201 310.5% sodium thiosulfate2On shaker32dH20Fast wash 33dH205 34Dehydrate 3 in each50% - 70% -95% - 100% - 100% ethanol 35Xylene x 23 in each 36Coverslip