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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
GREEN FLUORESCING PROTEIN
Dr. Osamu Shimomura
Aquorea victoria
Jelly fish
CONTAIN PHOTOORGANS OF A. victoria THAT
CONTAIN THE GREEN FLUORESCING PROTEIN
Excitation
Fluorescence
CONTAIN PHOTOORGANS OF A. victoria THAT
CONTAIN THE GREEN FLUORESCING PROTEIN
Mouse into which GFP gene has been introduced
Under reddish light
Under blue light
THE GREEN FLUORESCING PROTEIN
β-PLEATED SHEET BARREL
FLUOROPHORE IS SORT
STRETCH OF α-HELIX
HANGING IN THE INTERIOR
OF THE BARREL
EXCITATION
FLUORESCENCE
PEPTIDE LINKER
ESTROGEN RECEPTOR
GREEN FLUORESCING PROTEIN
PROTEIN OF
INTEREST
FLUORESCENCE
EXCITATION
GENE FOR THE PROTEIN OF INTEREST
NUCLEOTIDE SEQUENCE SPECIFYING
THE PEPTIDE LINKER
PLASMID
GFP GENE
AN EFFICIENT PROMOTER (e.g. the
promoter of the galactose operon of E. coli)
PLASMID “ORIGIN OF REPLICATION”
SEQUENCE (to allow replication of the plasmid
in the cell of interest)
MUTATIONS OF NORMAL GFP CAN CHANGE THE
EMISSION AND FLUORESCENCE SPECTRA
www.invitrogen.com/.../Image-Detail.9498.html
Live-cell imaging with Organelle Lights™ Endosomes-RFP and Mito-GFP in HeLa
cells. HeLa cells were transduced with Organelle Lights™ Endosomes-RFP and MitoRFP and co-stained with 1 mg/ml Hoechst 33342. Imaging was performed on live cells
using a Delta Vision Core microscope and standard DAPI/FITC/TRITC filter sets.
HIPPOCAMPAL CELL SPECIFICALLY
STAINED WITH GFP-MEMNBRANE
RECEPTOR PROTEIN
CELLULAR LIGHTS FROM INVITROGEN
Live-cell imaging with Organelle Lights™ Endosomes-RFP and Mito-GFP in HeLa
cells. HeLa cells were transduced with Organelle Lights™ Endosomes-RFP and MitoRFP and co-stained with 1 mg/ml Hoechst 33342 (nucleus) Imaging was performed on
live cells using a Delta Vision Core microscope and standard DAPI/FITC/TRITC filter sets.
www.invitrogen.com/.../Image-Detail.9498.html
Live-cell imaging with Cellular Lights™ Talin-GFP and Cellular Lights™ Talin-RFP. (A)
HeLa cells were transduced with Cellular Lights™ Talin-GFP and Actin-RFP. Imaging was
performed on live cells using a DeltaVision® Core microscope and standard FITC/TRITC filter
sets. (B) HeLa cells were transduced with Cellular Lights™ Talin-RFP and Organelle Lights™
Mito-GFP and co-stained with 1 µg/mL Hoechst stain for nuclei. Imaging was performed on live
cells using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filter sets.
Cells were transduced with Cellular Lights™ Histone
Cellular Lights™ MAP4-GFP
OUTER NUCLEAR MEMBRANE
NUCLEAR ENVELOPE
INNER NUCLEAR
MEMBRANE
NUCLEAR
PORE
NUCLEAR LAMINAR
NUCLEOLUS
CHROMOSOME
EUCHROMATIN
(CHROMOSOME IS THE
LEAST COMPACTED HER.
GENES CAN BE USED)
RIBOSOME
NUCLEAR PORE CONTAINING NUCLEAR
PORE COMPLEX
HETEROCHROMATIN
(CHROMOSOME VERY
COMPACTED HERE)
ROUGH
ENDOPLASMIC
RETICULUM