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Two-colour immunoenzymatic technique using sequential staining by APAAP to evaluate two cell antigens.
R Burgess1, K Hyde, P J Maguire
1University Department of Clinical Laboratory Haematology, Manchester Royal Infirmary, Oxford.
Journal of Clinical Pathology
|March 1, 1992
Summary
This study extends the alkaline phosphatase-antialkaline phosphatase (APAAP) method for double staining, enabling simultaneous detection of cell surface and nuclear antigens like bromodeoxyuridine (BrdU). The validated technique is useful for antigens in distinct cellular locations.
Area of Science:
- Immunohistochemistry
- Cell Biology
- Molecular Pathology
Background:
- The alkaline phosphatase-antialkaline phosphatase (APAAP) method is a widely used single staining technique.
- There is a need for methods that can detect multiple antigens simultaneously on the same slide.
- Detecting nuclear antigens alongside cell surface antigens presents unique technical challenges due to differing fixation requirements.
Purpose of the Study:
- To adapt the APAAP immunoenzyme single stain method into a versatile double stain technique.
- To enable the simultaneous evaluation of cell surface antigens and nuclear antigens, specifically bromodeoxyuridine (BrdU).
- To allow two primary antibodies of the same IgG isotype to be used for detecting distinct antigens.
Main Methods:
- Sequential application of the APAAP method using different dye couplings to a common alkaline phosphatase substrate.
- Production of distinct blue and red reaction products on the same slide to visualize two antigens.
- Utilizing differential fixation protocols, with initial fixation for surface antigens followed by alcohol/gluteraldehyde fixation for BrdU staining.
Main Results:
- The double staining technique successfully detected antigens on different cell populations and within different cellular compartments.
- Analysis of T lymphocyte subsets (CD4 and CD8) showed no significant difference in staining order or results compared to single stains.
- Discrete staining patterns were observed for antigens in different compartments, validating the technique's applicability.
Conclusions:
- The developed APAAP double staining technique is applicable for antigens with exclusive distribution and in different cellular compartments.
- It is particularly valuable for co-localizing antigens requiring different fixation methods, such as cell surface markers and DNA-incorporated BrdU.
- The method demonstrates a limited capacity for dual-color detection at a common antigen site.