Work procedure

this is the procedures for staining large sections.
A dish is used for staining large sections; however, a staining jar is used for staining standard-sized sections.

＊1 Rinsing

procedure
3 changes of distilled water Use plastic tweezers for the rest of this procedure.

＊2 Silver impregnation

procedure		section colors
Impregnating solution：0.5% silver protein solution overnight at 37℃
Wash the copper pieces with distilled water and gently submerge the pieces in silver protein aqueous solution.	※Place the copper pieces at the edges. ※When placing copper pieces into a staining jar with small sections, place the copper pieces at the edges or on the bottom.	After overnight immersion in a silver solution (light brown)

＊3 Washing & Reducing

procedure		section colors
Washing Wash in 3 changes of distilled water (bath 1, 2, and 3) Gently move the sections back and forth about five times in each bath. Do this process quickly. Reducing Reducing solution: hydroquinone and anhydrous sodium sulfate solution 10 minutes		Before immersion in reducing solution (light brown)
		After immersion in reducing solution (ocher)

＊4 Washing

procedure
tap water a few minutes

＊5 Rinsing ＆ Toning

procedure		section colors
Rinsing Wash in 3 changes of distilled water (bath 1, 2, and 3) Gently move the sections back and forth about five times in each bath. Toning Toning solution: 1% gold chloride solution 1 hour		Immediately after immersion in gold chloride (whitish) After 30 minutes, the color deepens the longer it is left in solution. After one hour, the color turns violet.

＊6 Washing

procedure
tap water 10 minutes Take care during this process because the stained color tone changes with the length of rinsing. Rinsing for longer than 10 minutes will cause the color tone to fade.

＊7 Reducing

procedure		section colors
2% oxalic acid solution 5 minutes Metal implements may be used from this point onwards.		Before immersion in 2% oxalic acid After immersion (deep purple)

＊8 Washing

procedure
tap water a few minutes

＊9 Fixing

procedure		section colors
Fixing：Photographic fixing solution (diluted 10 times) or 5% sodium thiosulfate aqueous solution 10 minutes The stained color tone changes with the length of time in the fixing solution. The longer the section is left in the solution, the sharper the contrast.		Before immersion in fixing solution After immersion (peony)

＊10 Washing

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

Result

Axon in the Internal capsule

Reagent

1. 0.5% silver protein solution
2. hydroquinone and anhydrous sodium sulfate solution
3. 1% gold chloride solution
4. 2% oxalic acid solution
5. Fixing：Photographic fixing solution (diluted 10 times) or 5% sodium thiosulfate aqueous solution

① 0.5% silver protein solution

Silver protein	0.5g
distilled water	100ml
metallic copper	5g

Sprinkle silver protein onto distilled water and allow it to dissolve from the surface downward. Do not stir because lather will form.	（Protargol S is used）
If fine particles float to the surface, then filter the solution. Pour gently to avoid creating lather. If lather forms, then push it to one corner with tweezers	（Protargol S is used）
Wash the copper pieces in distilled water. When placing the sections into the silver protein solution, gently submerge the copper pieces into the solution as well.	Albumosesilber is used

Note

• The silver protein aqueous solution concentration is 1% in the original protocol. However, adequate staining results can be obtained with a 0.5%-concentrated silver protein aqueous solution.
• The color tone of the stain differs depending on the silver protein manufacturer and between batches produced by the same manufacturer.

Protargol S Axon in the Internal capsule, stained with Protargol S.

Albumosesilber Axon in the Internal capsule, stained with Albumosesilber.

② Hydroquinone and anhydrous sodium sulfate solution

Dissolve hydroquinone in distilled water and then add anhydrous sodium sulfate.

Hydroquinone	1g
Anhydrous sodium sulfate solution	４g
distilled water	100ml

③ 1% gold chloride aqueous solution

This solution can be used repeatedly. Filter the solution after use and store it in a refrigerator.

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

Useful stock solutions

1% gold chloride solution （store in refrigerator）

2% oxalic acid solution

Fixing solution

Types of silver proteins

This is the silver protein used by the Laboratory of Neuropathology. Use caution because some products do not stain at all depending on the manufacturer. The products listed below are satisfactory for use.

product name	manufacturer	availability
Protargol ※ Caution	FUJIFILM Wako Pure Chemical Corporation	Available commercially
Albumosesilber	MERCK	out of stock
Albumosesilber DAB6	MERCK	out of stock
Protargol S	Daiichi Pharmaceutical Co., Ltd.	out of stock

※ Caution

The staining methods used for the Fujifilm Wako silver protein differ somewhat to the staining methods we use. Therefore, please refer to the Fujifilm Wako website when using their silver protein products.

Types of copper

The copper used in Bodian staining is not limited to copper pieces, and copper in various configurations can be used. Select a copper configuration that is easy to use. We use copper flakes from Kanto Chemical.