1. FLUORESCENCE MICROSCOPY

2. IMMUNOFLUORESCENCE

3. 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

4. BREAST OF A PREGNANT MAMMAL
DUCTULES
BREAST OF A PREGNANT MAMMAL

5. LOBULE
DUCTULE
DUCT
MILK PRODUCTION
MILK SECRETION

6. DUCTULE

7. NUCLEUS OF SECRETORY CELL

8. NUCLEUS OF MYOEPITHELIAL CELL

9. SEM OF BREAST ALVEOLUS (OR ACINUS)
SECRETORY CELL
MYOEPITHIAL CELL
FROM OUTSIDE ALVEOLUS
FROM INSIDE ALVEOLUS

10. BREAST SECRETORY CELL

11. 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

12. THE PLASMA MEMBRANE AROUND THE SECRETED FAT DROPLETS PREVENTS THEM FROM FLORMING CLUMPS IN THE MILK
BUTTER
BUTTER CHURN

16. FOR COMBINED IMAGE OF MULTIPLE FLUORESCENT ANTIBODIES (DIIFERENT COLOURED FLUOROPHORES):
PHOTOGRAPHS TAKEN WITH DIFFERENT EXCITATION LIGHTS MUST BE TAKEN AND THEN OVERLAPPED (“MERGED”)

17. 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)

21. 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
CANCEROUS BUT NOT YET INVASIVE
NON-CANCEROUS
INVASIVE CANCER

22. PEROXISOMES IN LIVER CELLS

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

24. BULL SPERM
DAPI

25. HeLA CELLS
NUCLEUS BLUE; GOLGI BODIES GREEN;MITOCHONDRIA RED

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

27. HeLa CELLS
Anti - ACTIN
DAPI
Anti - TUBILIN

28. Dr. Osamu Shimomura Aquorea victoria GREEN FLUORESCING PROTEIN
Jelly fish

29. CONTAIN PHOTOORGANS OF A
CONTAIN PHOTOORGANS OF A. victoria THAT CONTAIN THE GREEN FLUORESCING PROTEIN

30. Fluorescence
Excitation
CONTAIN PHOTOORGANS OF A. victoria THAT CONTAIN THE GREEN FLUORESCING PROTEIN

31. Mouse into which GFP gene has been introduced
Under reddish light
Under blue light

33. THE GREEN FLUORESCING PROTEIN
β-PLEATED SHEET BARREL
FLUOROPHORE IS SORT STRETCH OF α-HELIX HANGING IN THE INTERIOR OF THE BARREL

34. EXCITATION
FLUORESCENCE

36. PEPTIDE LINKER
ESTROGEN RECEPTOR
GREEN FLUORESCING PROTEIN

37. PROTEIN OF INTEREST
FLUORESCENCE
EXCITATION

38. PLASMID
GENE FOR THE PROTEIN OF INTEREST
NUCLEOTIDE SEQUENCE SPECIFYING THE PEPTIDE LINKER
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)

40. MUTATIONS OF NORMAL GFP CAN CHANGE THE EMISSION AND FLUORESCENCE SPECTRA

43.
Live-cell imaging with Organelle Lights™ Endosomes-RFP and Mito-GFP in HeLa cells. HeLa cells were transduced with Organelle Lights™ Endosomes-RFP and Mito-RFP and co-stained with 1 mg/ml Hoechst Imaging was performed on live cells using a Delta Vision Core microscope and standard DAPI/FITC/TRITC filter sets.

44. HIPPOCAMPAL CELL SPECIFICALLY STAINED WITH GFP-MEMNBRANE RECEPTOR PROTEIN

45. CELLULAR LIGHTS FROM INVITROGEN

47. Live-cell imaging with Organelle Lights™ Endosomes-RFP and Mito-GFP in HeLa cells. HeLa cells were transduced with Organelle Lights™ Endosomes-RFP and Mito-RFP and co-stained with 1 mg/ml Hoechst (nucleus) Imaging was performed on live cells using a Delta Vision Core microscope and standard DAPI/FITC/TRITC filter sets.

49. 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.

51. Cells were transduced with Cellular Lights™ Histone
Cellular Lights™ MAP4-GFP

52. NUCLEOLUS OUTER NUCLEAR MEMBRANE NUCLEAR ENVELOPE
INNER NUCLEAR MEMBRANE
NUCLEAR ENVELOPE
CHROMOSOME
NUCLEOLUS
NUCLEAR PORE
NUCLEAR PORE CONTAINING NUCLEAR PORE COMPLEX
ROUGH ENDOPLASMIC RETICULUM
RIBOSOME
HETEROCHROMATIN
(CHROMOSOME VERY COMPACTED HERE)
EUCHROMATIN
(CHROMOSOME IS THE LEAST COMPACTED HER. GENES CAN BE USED)
NUCLEAR LAMINAR