Make 4% of  NaOH (sodium hydroxide) - use gloves, store in fume hood

Make 4% of NaOH (sodium hydroxide) – sodium hydroxide – this is in the Kentros lab make in fume hood, 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

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

Make pyridine + acetic acid - use gloves, store in fume hood

100ml pyridine + 50ml acetic acid

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

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)

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

A	B	C	D
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