FLUORESCENCE MICROSCOPY

IMMUNOFLUORESCENCE

Multiple immunofluorescence labelling of formalin-fixedparaffin-embedded (FFPE) tissueDavid 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

BREAST OF A PREGNANT MAMMAL

DUCTULES

LOBULE

DUCTULE

DUCT

MILK PRODUCTION

MILK SECRETION

DUCTULE

NUCLEUS OF SECRETORY CELL

NUCLEUS OF MYOEPITHELIAL CELL

SEM OF BREAST ALVEOLUS (OR ACINUS)

FROM OUTSIDE ALVEOLUS FROM INSIDE ALVEOLUS

SECRETORY CELL

MYOEPITHIAL CELL

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 CHURNBUTTER

FOR COMBINED IMAGE OF MULTIPLE FLUORESCENT ANTIBODIES (DIIFERENT COLOURED FLUOROPHORES):

PHOTOGRAPHS TAKEN WITH DIFFERENT EXCITATION LIGHTS MUST BE TAKEN AND THEN OVERLAPPED (“MERGED”)

LUMEN OF DUCTULE

MYOEPITHELIAL CELL

INTERMEDIATE FILAMENTS OF SECRETORY CELLS

NUCLEUS OF SECRETORY CELL

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

NON-CANCEROUS CANCEROUS BUT NOT YET INVASIVE

INVASIVE CANCER

ANTIBODY AGAINST ESTROGEN RECEPTOR

ANTIBODY AGAINST SECRETORY CELL KERATINS

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

Anti - TUBILINAnti - ACTIN DAPI

HeLa CELLS