LIFE 212 Lecture Notes - Fibroblast, Immunofluorescence, Mitochondrion
30 Mar 2014
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Document Summary
Pellet (700 x g) + mb chloroplasts. Supernatant (700 x g) + mb mitochondria. Chloroplasts: dcpip acts as a final electron acceptor: gets reduced directly by. Mitochondria: mb gets reduced indirectly by the depletion of oxygen from the media in the presence of active mitochondrial etc/qualitative assay. Can co-stain multiple structures in the same cell. Direct staining: fluorescent dye (fluorochrome) directly binds to cellular components. Flurophore conjugated to a probe that binds to specific structures e. g. phalloidin. Gfp gene is fused to gene of interest, allows live cell imaging. Absorb at one wl specific to the fluorophore and emit at another specific wavelength. Excitation filter: filters light wavelengths allowing only specific absorbing wavelength (excitation) to illuminate the dye in the specimen. Emission filter: allows only the emitted light (emission) to reach the eye, dye glows against a dark background. This week"s experiment: fluorescence staining of 3t3l1 fibroblast cells. 2 antibody fluorophore conjugate + hoechst + phalloidin-alexa-fluor488.
