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.
|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|
|6||Antigen retrieval||0.01M citric acid buffer solution, pH6.0||Autoclave for 10 minutes at 120℃|
|7||Washing||Running tap 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|
|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|
※ PBST is not used from ⑪ onward because it weakens the autofluorescence reduction effect.
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.
※ Some antibodies such as Aβ require formic acid treatment.
※ Use sera and secondary antibodies from the same animal species.