⑫ Staining

Immunohistochemical staining：Enzyme antibody technique

The immunohistochemical staining is used to detect a specific protein by using an antigen-antibody reaction. In this method, enzymes are used for visualizing the antigen-antibody complex.

• Staining procedure
• Detection method
• Antigen retrieval
• Reagents used for this procedure

Staining procedure

Polymer method with enzyme peroxidase marker

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	Washing	Running tap water	2 minutes
5	Rinsing	Distilled water
6	Blocking endogenous peroxidase	3% hydrogen peroxide solution	15 minutes
7	Washing	Running tap water
8	Rinsing	Distilled water
9	Antigen retrieval	0.01M citric acid buffer solution, pH6.0	Autoclave for 10 minutes at 120℃
10	Washing	Running tap water
11	Rinsing	Distilled water
12	Washing	PBS	3 changes, 5 minutes each
13	Serum blocking	10% normal serum + 0.01% sodium azide solution	15 minutes to more than 1 hour at RT
14	Primary antibody		Overnight, 4℃
15	Washing	PBS	3 changes, 5 minutes each
16	Secondary antibody	HRP-polymer secondary antibody	RT
17	Washing	PBS	3 changes, 5 minutes each
18	Developping	DAB substrate	Approximately 1 to 15 minutes, RT
19	Washing	Running tap water
20	Rinsing	Distilled water
21	Counterstaining	Hematoxylin solution	1 minutes
22	Blueing	Running tap water	15 minutes
23	Dehydration	70%, 95% ethanol	5 minutes each
24	Dehydration	100% ehtanol	3 changes, 5 minutes each
25	Clearing	Xylene	3 changes, 10 minuntes each
26	Coverslipping

Detection method

Various methods are used to detect the antigen-antibody reaction, including the direct, ABC, LSAB, and polymer methods.

Various kits for each detection method are available commercially. Here, I will introduce the kits used in the Laboratory of Neuropathology.

① ABC method (ABC kit, VECTOR LABORATORIES, INC.)

There are three types of marker enzymes, comprising peroxidase, alkaline phosphatase, and glucose oxidase.

Mechanisms of the ABC method

• The primary antibody reacts with the antigen.
• The biotinylated second antibody reacts with the primary antibody.
• The avidin-biotin-peroxidase complex reacts with the secondary antibody.
• The peroidase substrates, such as DAB, react with the enzyme label, and visualize the target protein.

② Labeled streptavidin biotin (LSAB) method (SAB-PO kit, NICHIREI BIOSCIENCES INC.)

The marker enzymes are peroxidase and alkaline phosphatase.

Mechanisms of the LSAB method

• The primary antibody reacts with the antigen.
• The biotinylated secondary antibody reacts with the primary antigen.
• The streptavidin-enzyme conjugate reacts with the secondary antibody.
• The peroidase substrates, such as DAB, react with the enzyme label, and visualize the target protein.

③ HRP-Polymer method (EnVision Plus Kit, Agilent Technologies, Inc.)

The marker enzymes are peroxidase and alkaline phosphatase.

Mechanisms of the polymer system

• The primary antibody reacts with the antigen.
• The polymer-enzyme complex reacts with the primary antibody.
• The peroidase substrates, such as DAB, react with the enzyme label, and visualize the target protein.

Antigen retrieval

The antigenic sites are masked by formalin fixation and may not be able to bind with the antibody. Therefore, the masked antigenic sites are exposed by antigen retrieval processing and are then able to bind with the antibody.

Antigen retrieval methods include heat treatment, proteolytic enzyme processing, and formic acid treatment.

Heat treatment

① Microwave oven

1. Place slides in a heat-resistant container filled with the buffer. Lightly cover the container with plastic wrap.
2. Add distilled water to another container.
3. Heat the containers in a domestic microwave oven for 10 minutes.
4. If the buffer evaporates and the slides come out from the water's surface, then add the distilled water that was heated at the same time.
5. Heat for an additional 5 minutes.
6. Leave the container at room temperature to cool. Once it is cool enough to touch, remove the slides and wash them with tap water.

② Autoclave

1. Place slides in a heat-resistant container filled with the buffer, and cover the container with a lid.
2. Heat in an autoclave for 10 minutes at 120 °C.

Buffer solutions for heal treatment

• 0.01M Citric acid buffer solution、pH6.0、pH7.0、pH8.0
• 1mM EDTA、pH8.0

Proteolytic processing

• 0.04% proteinase K solution for 10minutes at room temperature.
• 0.1% trypsin for 30min at 37℃

Formic acid treatment

• Place the formic acid stock solution on the section for five minutes at room temperature.

※ Some antibodies such as Aβ require formic acid treatment.

Reagents used for this procedure

① washing solution

• PBS
• PBST (PBS + 0.05%Tween20)

② Blocking solution for quenching endogenous peroxidase activity

• 3% hydrogen peroxide solution

③ Antigen retrieval solution

• See the antigen retrieval section

④ Blocking solution

• 10% normal serum + 0.01% sodium azide solution

※ Use sera and secondary antibodies from the same animal species.

⑤ Enzyme substrate ( for the peroxidase detection system)

• Add 5μl of 30% hydrogen peroxide to 5mg of 3,3' diaminobenzidine (DAB) in 10ml of PBS