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FLUORESCENCE MICROSCOPY IMMUNOFLUORESCENCE Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue David Robertson, Kay Savage, Jorge S Reis-Filho and Clare M Isacke* Address: Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK PUBLISHED 2008 DUCTULES BREAST OF A PREGNANT MAMMAL MILK PRODUCTION LOBULE DUCTULE DUCT MILK SECRETION DUCTULE NUCLEUS OF SECRETORY CELL NUCLEUS OF MYOEPITHELIAL CELL SEM OF BREAST ALVEOLUS (OR ACINUS) MYOEPITHIAL CELL SECRETORY CELL FROM OUTSIDE ALVEOLUS FROM INSIDE ALVEOLUS BREAST SECRETORY CELL LIPID DROPLETS BEING SECRETED FROM THE SECRETORY CELLS OF BREAST DUCTULE EPITHELIUM LIPID DROPLET BEING EXTRUDED (IT IS COVERED IN PLASMA MEMBRANE) SECRETORY CELL MICROVILLI THE PLASMA MEMBRANE AROUND THE SECRETED FAT DROPLETS PREVENTS THEM FROM FLORMING CLUMPS IN THE MILK BUTTER CHURN BUTTER FOR COMBINED IMAGE OF MULTIPLE FLUORESCENT ANTIBODIES (DIIFERENT COLOURED FLUOROPHORES): PHOTOGRAPHS TAKEN WITH DIFFERENT EXCITATION LIGHTS MUST BE TAKEN AND THEN OVERLAPPED (“MERGED”) MYOEPITHELIAL CELL LUMEN OF DUCTULE NUCLEUS OF SECRETORY CELL INTERMEDIATE FILAMENTS OF SECRETORY CELLS ANTI- SMOOTH MUSCLE ACTIN ANTI-GLANDULAR CELL KERATIN DAPI (DNA-STAINING) DETECTION OF BREAST CANCER BY IMMUNOFLUORESCENCE THIS PARTICULAR CANCER IS DUE TO UNCONTROLLED GROWTH OF THE EPITHELIAL CELLS OF THE MILK DUCTS ANTIBODY AGAINST ESTROGEN RECEPTOR ANTIBODY AGAINST SECRETORY CELL KERATINS NON-CANCEROUS CANCEROUS BUT NOT YET INVASIVE INVASIVE CANCER PEROXISOMES IN LIVER CELLS HeLA CELLS DAPI notice light and darker staining areas in the nucleus (HETEROCHROMATIN / EUCHROMATIN) Anti-ACTIN ANTIBODY BULL SPERM DAPI HeLA CELLS NUCLEUS BLUE; GOLGI BODIES GREEN;MITOCHONDRIA RED RAT URINARY EPITHELIAL CELLS Anti- CYTOCHROME OXIDASE ANTIBODY Anti- ACTIN ANTIBODY DAPI HeLa CELLS Anti - ACTIN Anti - TUBILIN DAPI