A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays
| Autor: | Felipe A. Beltrán, Abraham Rosas-Arellano, Juan B. Villalobos-González, Fanis Missirlis, Maite A. Castro, Lourdes Palma-Tirado, Alfonso Cárabez-Trejo |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Histology Fluorescent Antibody Technique Biology Immunofluorescence Epitope Antibodies 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine medicine Animals Hydrogen peroxide Microscopy Immunoelectron Molecular Biology Fixative medicine.diagnostic_test Cell Biology Immunogold labelling Molecular biology Mice Inbred C57BL Medical Laboratory Technology 030104 developmental biology chemistry Glycine Immunohistochemistry 030217 neurology & neurosurgery Immunostaining |
| Zdroj: | Histochemistry and cell biology. 146(4) |
| ISSN: | 1432-119X |
| Popis: | Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods. |
| Databáze: | OpenAIRE |
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