⑫ Staining

Immunohistochemical staining:Fluorescent staining method

The immunostaining technique is a staining method that uses an antigen-antibody reaction in which an antibody binds to an antigen.

Antibodies that bind to specific antigens are reacted on the section, and the resulting antigen-antibody complex is stained and visualized.

Staining methods that use fluorescent dye for structure visualization are called fluorescent staining methods.

Staining procedure

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6Antigen retrieval0.01M citric acid buffer solution, pH6.0Autoclave for 10 minutes at 120℃
7WashingRunning tap water 
8RinsingDistilled water 
9WashingPBS3 changes, 5 minutes each
10Serum blocking10% normal serum + 0.01% sodium azide solution15 minutes to more than 1 hour at RT
11Primary antibody Overnight, 4℃
12WashingPBS3 changes, 5 minutes each
13Rinsing70% ethanolQuckly
14Quenching autofluorescenceTrueBlack® Lipofuscin Autofluorescence Quencher3 minutes, RT
15WashingPBS3 changes, 5 minutes each
16Secondary antibody 1 hour, RT
17WashingPBS3 changes, 5 minutes each
18RinsingPBS + 0.02%sodium azide solution 

※ PBST is not used from ⑪ onward because it weakens the autofluorescence reduction effect.

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 Antigen retrieval solution

③ Blocking solution

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

④ Reagent for quenching lipofuscin autofluorescence