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
• Antigen retrieval
• Reagents used for this procedure

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	Washing	Running tap water	2 minutes
5	Rinsing	Distilled water
6	Antigen retrieval	0.01M citric acid buffer solution, pH6.0	Autoclave for 10 minutes at 120℃
7	Washing	Running tap water
8	Rinsing	Distilled water
9	Washing	PBS	3 changes, 5 minutes each
10	Serum blocking	10% normal serum + 0.01% sodium azide solution	15 minutes to more than 1 hour at RT
11	Primary antibody		Overnight, 4℃
12	Washing	PBS	3 changes, 5 minutes each
13	Rinsing	70% ethanol	Quckly
14	Quenching autofluorescence	TrueBlack® Lipofuscin Autofluorescence Quencher	3 minutes, RT
15	Washing	PBS	3 changes, 5 minutes each
16	Secondary antibody		1 hour, RT
17	Washing	PBS	3 changes, 5 minutes each
18	Rinsing	PBS + 0.02%sodium azide solution
19	Coverslipping

※ 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

• 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 Antigen retrieval solution

• Refer to the antigen activation section

③ Blocking solution

• 10% normal serum + 0.01% sodium azide solution

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

④ Reagent for quenching lipofuscin autofluorescence

• 0.1% Sudan black B solution in 70% ethanol

or

• TrueBlack® Lipofuscin Autofluorescence Quencher, 20X in DMF（Available commercially）