Write a 7 pages paper on lab report practical fluorescence microscopy practical. The PFA fixed cells were permeabilised using triton-x 100 (TX 100). Cells were then incubated with the primary antibody, against the target protein, and washed thoroughly to remove the unbound antibodies. Then the cells were incubated with secondary antibody targeted against the primary antibody, and again washed with PBS. Cell nuclei were counter stained with Hoechst33342. Coverslips with the cells were mounted with Mowiol on clean glass slide and observed under a fluorescence microscope after hardening of the Mowiol. Using proper filters and the objectives, the target proteins were viewed and images were taken and stored. Specific acquisition parameters of the imagery were noted for the record. Introduction: Fluorescence microscopy utilizes two facts: one that an antibody binds to an antigen and second, that fluorescent dyes or fluorochromes emit light of a longer wavelength when irradiated with short wavelength light (Herman, 1997). Only some proteins (Mocz, 1999) have this inherent property possessed by fluorochromes. others have to be rendered fluorescent by tagging them with fluorochromes. For this purpose the protein of interest is first bound by a primary antibody against that protein and then a secondary antibody tagged with a fluorochrome is bound to the primary antibody.
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