Application and classification of ELISA kits in cellular immunofluorescence - Master's thesis - Dissertation

Before incubating the primary antibody, it's important to turn off the incubation timer and avoid washing the sample before adding the primary antibody. Only remove any remaining liquid gently. After that, there's no need to close the chamber again. However, both the primary and secondary antibodies should be diluted in a BSA-rich buffer solution to reduce non-specific binding. Some researchers also add Tween-20 to further block non-specific interactions. Another common practice is to use serum from the same animal species as the secondary antibody for blocking, which can help improve signal specificity. Immunofluorescence is a powerful technique used in biological research, often referred to as fluorescent antibody technology. It was one of the earliest methods developed for detecting antigens or haptens within cells. This method relies on the use of fluorescently labeled antibodies to bind specifically to target antigens, allowing for precise localization under a fluorescence microscope. When performing immunofluorescence, especially in double-labeling experiments where two antigens are being detected simultaneously on the same cell or tissue, two layers of fluorescent staining are typically required. These can be carried out using either a direct or indirect labeling approach. The process involves several key steps: preparing cell smears, fixing and permeabilizing the cells, blocking non-specific sites, incubating with primary and secondary antibodies, and finally visualizing the fluorescent signals. Common challenges in immunofluorescence include issues like high background noise, weak signals, and improper localization. Proper optimization of antibody concentrations, blocking agents, and incubation times can significantly improve results. Additionally, the technique is widely used in developmental biology, such as studying the localization of the epidermal growth factor receptor during mouse embryo development. There are several types of immunosymbol techniques, including fluorescence immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), biotin-avidin system, time-resolved fluorescence immunoassay, and dissociation-enhanced lanthanide fluorescence immunoassay. Each has its own advantages depending on the application and sensitivity requirements.

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