Triton X-100 pretreatment of LR-white thin sections improves immunofluorescence specificity and intensity
| Autor: | Salem Ghrebi, Donald M. Brunette, Gethin Owen |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Antigenicity
Histology Plastic Embedding Octoxynol Preservation Biological Acrylic Resins Fluorescent Antibody Technique Immunofluorescence Sensitivity and Specificity law.invention Cell Line chemistry.chemical_compound Immunolabeling Antigen law Microtome medicine Animals Instrumentation Chromatography medicine.diagnostic_test Chemistry Macrophages Microtomy Immunohistochemistry Staining Rats Medical Laboratory Technology Triton X-100 Microscopy Electron Scanning Anatomy Immunostaining Cryoultramicrotomy |
| Zdroj: | Microscopy research and technique. 70(7) |
| ISSN: | 1059-910X |
| Popis: | The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired. |
| Databáze: | OpenAIRE |
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