Work procedure

＊1 Oxidizing

procedure		section colors
0.25% potassium permanganate solution 15 minute		Turns reddish brown

＊2 Decolorizing

procedure		section colors
2% oxalic acid solution 1 minute		Before immersion in oxalic acid After immersion (white)

＊3 Washing ＆ Silver impregnation ＆ Rinsing

procedure
Washing Distilled water 1 minutes × 3 times Use plastic tweezers from this point on. Silver impregnation Alkaline silver iodide solution 2 〜 5 minute Rinsing 0.5% acetic acid solution 1 minutes × 3 times

＊4 Reducing ＆ Rinsing

procedure		section colors
Reducing Use cold solution to slow the reaction. React until the section is stained a pale color that enables visualization of the structures within the entire section. React in a dish to see the color change. The reaction time is often longer than 10 minutes; however, be aware that the reaction time differs depending on the section. ※Section A：Changes color in five minutes and is complete in 15 minutes ※Section B：Changes color in 13 minutes and is complete in 17 minutes If you perform the reaction using reducing solution at room temperature, then the reaction will progress extremely quickly and be impossible to control. Therefore, always use a chilled reducing solution. Rinsing 0.5% acetic acid solution 1 minutes × 3 times	The reducing solution will darken over time. If the liquid is left in a glass container, then it will permanently stain the container due to the silver mirror reaction. Therefore, containers must be immediately washed after use.	Section A After 8 minutes：Some parts of the section turn a very faint blackish brown After 15 minutes：All of the structures are blackish brown in color After 20 minutes：The entire section is a blackish brown color. The overall structures are difficult to discern. Section B After 13 minutes：Some parts of the section turn a very faint blackish brown After 17 minutes：All of the structures are a blackish brown color

＊5 Rinsing ＆ Toning

procedure		section colors
Rinsing Distilled water 1 minutes × 3 times Toning 1% gold chloride solution 5 〜 15 minutes		Reduction 8 minutes：Turns white Reduction 15 minutes：Turns gray. The excess color fades, and the contours become sharp. Reduction 20 minutes：Turns blackish gray

＊6 Washing

procedure
Running tap water A few minutes Metal implements may be used from this point on.

＊7 Fixing

procedure		section colors
Photographic fixing solution (diluted 10 times) or 5% sodium thiosulfate aqueous solution 10 minutes		Reduction 8 minutes：Very faint lilac Reduction 15 minutes：Lilac Reduction 20 minutes：Gray purple

＊8 Washing

procedure
Running tap water Wash more than 10 minute, otherwise the section color will change

＊9 Counterstaining

procedure
Counterstain in Kernechtrot 5 minutes

Staining results

The staining results differ depending on the reduction time.

Reduction time of 8 minutes	Reduction time of 15 minutes	Reduction time of 20 minutes
40 times 200 times 400 times	40 times 200 times 400 times	40 times 200 times 400 times

Reduction time of 8 minutes

The overall color is faint. This will not produce a positive staining image.

Reduction time of 15 minutes

The section is stained a sharp black, and the shapes of fine fibers can be discerned.

Reduction time of 20 minutes

The background is stained black. The cells are stained completely black, making it impossible to discern detailed structures.

Issues to consider with reducing solution reactions

Temperature of the reducing solution

Performing a reaction using room-temperature reducing solution will result in the reaction progressing too quickly, and the entire section will have a blackish brown stain.

To control the reaction, conduct it slowly using chilled reducing solution.

Chilled reducing solution：After five minutes

Room temperature reducing solution：After five minutes

Types of glass slide

With MAS-coated glass slides, the entire glass turns black. Therefore, you should avoid using this type of glass; however, the pathological deposits can be detected.

We use silane coating or egg white albumin coating.

Chilled reducing solution：After five minutes

Reagent

1. 0.25% potassium permanganate solution
2. 2% oxalic acid solution
3. Alkaline silver iodide solution
4. 0.5% acetic acid solution
5. Reducing solution (mixture of solutions A and B)
6. 1% gold chloride solution
7. Photographic fixing solution (diluted 10 times)

③ Alkaline silver iodide solution

Dissolve sodium hydroxide and potassium iodide in that order in distilled water and then mix slowly with 1% silver nitrate aqueous solution.

The solution becomes cloudy when the silver nitrate is added; however, but it will become clear once everything is mixed together.

sodium hydroxide	４g
Potassium iodide	10g
1% silver nitrate solution	3.5ml
distilled water	100ml

⑤ Reducing solution (mixture of solutions A and B)

solutions A

Dissolve ammonium nitrate, silver nitrate, and tungstosilicic acid in that order in distilled water and add aqueous formaldehyde solution afterwards.

ammonium nitrate	0.2g
silver nitrate	0.2g
Tungstosilicic acid	1g
Formaldehyde solution (37% to 38%)	0.5ml
distilled water	100ml

solutions B

Dissolve anhydrous sodium carbonate in distilled water.

Anhydrous sodium carbonate	5g
distilled water	100ml

Reducing solution

Pour small amount of solution A into solution B at a time until equal parts of solutions A and B are mixed. The mixture is best prepared fresh each time. The solution will become cloudy when solution A is added; however, it will become clear once the solutions are mixed together.

1% gold chloride solution

This solution can be used repeatedly. Filter the solution after use and then refrigerate it.

Gold(Ⅲ) chloride acid tetrahydrate	１g
distilled water	100ml

Quick tips for reagents

Some reagents are stable and may be used repeatedly.

Convenient reagents to prepare in advance

Reducing solution A and B (Store refrigerated. )	Alkaline silver iodide solution (store in refrigerator)	1% silver nitrate solution (store in refrigerator)   ※ Please note that reagents will turn black if they are stored for a long period. Discard it when it turns black.
1% gold chloride solution (store in refrigerator)	2% oxalic acid solution	Fixing solution

※ 1% gold chloride and 2% oxalic acid aqueous solutions as well as fixing solution can be stored for a long time and used for other silver stainings. Therefore, it is convenient to always have these reagents prepared.

Reagents that can be used multiple times (Store refrigerated.)

There are reagents that can be reused at a later date after previous use.

① Reagents that can be reused for staining over consecutive days

• 0.25% potassium permanganate aqueous solution: Can be reused several times. However, it changes color when stored for a long period. Therefore, it should be discarded after approximately one week.
• )2% oxalic acid aqueous solution：Can be reused once or twice; however, reuse depends on the number of slides stained at one time.

② Reagents that can be repeatedly reused over a long period of time

• Alkaline silver iodide solution: Filter after use. Discard the solution when it becomes cloudy.
• 1% gold chloride aqueous solution: Filter after use. This solution lasts a long time.