⑫ Staining

Holzer staining

This method stains fibrous astrocytic processes blue purple with crystal violet. The proliferation of astrocyte fibrous components in response to tissue damage and gliosis can be observed topographically.

Staining procedure

1	Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95%, 70% ethanol	5 minutes each
4	Mordanting ＊1	Phosphomolybdic alcohol	1 minute
5	Morderting ＊2	Chloroform-alcohol mixture	Blot with the filter paper (four-ply) damped with chloroform-alcohol mixture, and flood with the mixture. Leave the section until it becomes transparent (around 10 seconds)
6	Staining ＊3	2% crystal violet solution	Drain the chloroform-alcohol mixture and replace with the crystal violet solution. Discard the solution and blot well with filter paper (four-ply).
7	Washing ＊4	10% pottasium bromide solution	More than 1 minute
8	Differentiation ＊5	Aniline-xylene mixture	Blot with the filter paper (four-ply) damped with aniline-xylene mixture, and flood with the mixture. Change the liquid approximately every 15 minutes.
9	Microscopic check		Continue differentiation until only the glia fibers appear stained.
10	Washing ＊6	Xylene	2 changes, 1 minutes each
11	Clearing ＊6	Xylene	3 changes, 10 minuntes each
12	Coverslipping ＊6

Work procedure

③ 〜 ⑥ Experiment video

※1 Mordanting

procedure
Phosphomolybdic alcohol one minute

procedure

Phosphomolybdic alcohol

one minute

※2 Morderting

procedure
chloroform-alcohol mixture Damp the filter paper (4-ply) with the mixture.
Blot well with the filter paper. Press down evenly and strongly.
Flood with the mixture without allowing sections to dry. Leave the section until it becomes transparent (around 10 seconds). ※ The chloroform-alcohol mixture evaporates rapidly. Therefore, the liquid can be refilled.

procedure

chloroform-alcohol mixture

Damp the filter paper (4-ply) with the mixture.

Blot well with the filter paper.
Press down evenly and strongly.

Flood with the mixture without allowing sections to dry.
Leave the section until it becomes transparent (around 10 seconds).

※ The chloroform-alcohol mixture evaporates rapidly. Therefore, the liquid can be refilled.

※3 Staining

procedure
2% crystal violet solution Immediately after draining the chloroform-alcohol mixture, replace with the crystal violet solution.
Discard the crystal violet solution. Blot well with filter paper (4-ply). Press down evenly and strongly.

procedure

2% crystal violet solution

Immediately after draining the chloroform-alcohol mixture, replace with the crystal violet solution.

Discard the crystal violet solution.
Blot well with filter paper (4-ply).
Press down evenly and strongly.

※4 Washing

procedure
10% potassium bromide solution Apply 10% potassium bromide solution
Leave the section for at least one minute.

procedure

10% potassium bromide solution

Apply 10% potassium bromide solution

Leave the section for at least one minute.

※5 Differentiation

procedure

aniline-xylene mixture

Damp the filter paper (4-ply) with the aniline-xylene mixture.

Blot the filter paper.
Press down evenly and strongly.

Flood the aniline-xylene mixture and then leave the section.

Change the aniline-xylene mixture approximately every 15 minutes.

※ The liquid does not need to be changed frequently.

Differences in the staining results depending on the differentiation time

After 15 minutes of differentiation	After 30 minutes of differentiation	After 45 minutes of differentiation

After checking with a microscope as needed to determine whether there is sufficient differentiation, discard the aniline-xylene mixture on the section and apply xylene.
After leaving the section for some time, discard the xylene and apply new xylene.

※ Wash the tweezers in xylene.