FLUORESCENCE MICROSCOPY IMMUNOFLUORESCENCE Multiple immunofluorescence labelling of formalinfixed

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 SW 3 6 JB, UK PUBLISHED 2008

DUCTULES BREAST OF A PREGNANT MAMMAL

MILK PRODUCTION LOBULE 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 BUT NOT YET INVASIVE CANCER

PEROXISOMES IN LIVER CELLS

He. LA CELLS DAPI notice light and darker staining areas in the nucleus (HETEROCHROMATIN / EUCHROMATIN) Anti-ACTIN ANTIBODY

BULL SPERM DAPI

He. LA CELLS NUCLEUS BLUE; GOLGI BODIES GREEN; MITOCHONDRIA RED

RAT URINARY EPITHELIAL CELLS Anti- CYTOCHROME OXIDASE ANTIBODY Anti- ACTIN ANTIBODY DAPI

He. La CELLS Anti - ACTIN Anti - TUBILIN DAPI