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

Polymer method with enzyme peroxidase marker

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6Blocking endogenous peroxidase3% hydrogen peroxide solution15 minutes
7WashingRunning tap water 
8RinsingDistilled water 
9Antigen retrieval0.01M citric acid buffer solution, pH6.0Autoclave for 10 minutes at 120℃
10WashingRunning tap water 
11RinsingDistilled water 
12WashingPBS3 changes, 5 minutes each
13Serum blocking10% normal serum + 0.01% sodium azide solution15 minutes to more than 1 hour at RT
14Primary antibody Overnight, 4℃
15WashingPBS3 changes, 5 minutes each
16Secondary antibodyHRP-polymer secondary antibodyRT
17WashingPBS3 changes, 5 minutes each
18DeveloppingDAB substrateApproximately 1 to 15 minutes, RT
19WashingRunning tap water 
20RinsingDistilled water 
21CounterstainingHematoxylin solution1 minutes
22BlueingRunning tap water15 minutes
23Dehydration70%, 95% ethanol5 minutes each
24Dehydration100% ehtanol3 changes, 5 minutes each
25ClearingXylene3 changes, 10 minuntes each

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.


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

Mechanisms of the ABC method

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

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

The marker enzymes are peroxidase and alkaline phosphatase.

Mechanisms of the polymer system

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

Proteolytic processing

Formic acid treatment

※ Some antibodies such as Aβ require formic acid treatment.

Reagents used for this procedure

① washing solution

② Blocking solution for quenching endogenous peroxidase activity

③ Antigen retrieval solution

④ Blocking solution

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

⑤ Enzyme substrate ( for the peroxidase detection system)