Gallyas-silver stain

Christiana.bjorkli; María José Lagartos Donate

Feb 15, 2022

Gallyas-silver stain

DOI

dx.doi.org/10.17504/protocols.io.b44cqysw

Christiana.bjorkli¹,
María José Lagartos Donate²

¹The Norwegian University of Science and Technology;
²University of Oslo

Sandvig lab

Christiana.bjorkli

DOI: dx.doi.org/10.17504/protocols.io.b44cqysw

Protocol Citation: Christiana.bjorkli, María José 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

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

5%: 3ml acetic acid in 597ml H20
1%: 0.6ml acetic acid in 600ml H20
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

 
StepReagent Minutes Comments 
1dH203 or morePut in dH20 while preparing other solutions
2Pyridine + acetic acid60On shaker
350% 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 nitrate
8Silver nitrate60On shaker – make A+ B + C
90.5% acetic acid 10 
10A + B + C6 (15min human)Do not let the tissue become dark, time is dependent on sample
110.5% acetic acid1New
120.2% 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 step
19A + B + C3-4On shaker
200.5% acetic acid Use the one from the previous step
210.2% potassium ferricyanide3On shaker 
22dH201 
230.5% sodium thiosulfate2On shaker
24dH204 
25dH204 
260.5% acetic acid1New, can be reused
27A + B + C3-4On shaker
280.5% acetic acid1Use the one from the previous step
290.2% potassium ferricyanide10On shaker
30dH201 
310.5% sodium thiosulfate2On shaker
32dH20Fast wash 
33dH205 
34Dehydrate 3 in each50% - 70% -95% - 100% - 100% ethanol 
35Xylene x 23 in each 
36Coverslip   
 


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

Public workspaceGallyas-silver stain

Gallyas-silver stain