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Emma Lundberg
Marie Skogs (technician), Charlotte Stadler (PhD-student), Anna Stenius (technician), Ulla Wrethagen (technician)
To analyze the subcellular distribution of proteins, including; (i) immunofluorescent staining of cell lines, (ii) confocal imaging of immunofluorescently stained samples, (iii) annotation of subcellular staining pattern.
The immunofluorescence module is responsible for the analysis of subcellular localizations of proteins using antibodies generated in the Human Protein Atlas (HPA) program.
Three cell lines are cultured in vitro, fixated/permeabilized using formaldehyde/detergent treatment and immunofluorescently stained according to standardized protocols. Besides the HPA antibodies, the cells are also stained with two organelle probes specific for the endoplasmatic reticulum (ER) and micro-tubules, as well as counterstained with the nuclear probe DAPI.
Two high resolution, multicolor images are acquired per antibody and cell line. The images are acquired manually using a Zeiss LSM 510 Meta, confocal laser scanning microscope equipped with a 63x magnification, oil immersion objective. The microscope operator ensures that the acquired images are representative for the entire sample and positions the optical section at an appropriate level in the cells.
All images are manually annotated using a web-based annotation document. For each image pair the subcellular localization, characteristics and intensity of the staining is described.
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