1. Lesson 10 Fluorescence Staining =Fluorescent labeling

2. Lecture overview Fluorescence (What? / Why?)
Fluorescence Staining (How?)
Types & Examples

3. Why fluorescence?
Contrast!
Brilliant signals against dark background

4. Why Fluorescence staining ?
widely used approaches for subcellular compartments and localizing proteins
Nonimmunological Fluorescent labeling
Immunological Fluorescent labeling
(=Immunofluorescence)

5. Fluorescent stain
Fluorescent compounds (probe) may be used to directly label specific subcellular components and macromolecules.

6. Fluorescent stain
Fluorescent compounds (probe) may be used to directly label specific subcellular components and macromolecules.
Fluorescent compounds (probe) may be used to indirectly label specific subcellular components and macromolecules.
Coupling fluorescent compounds to molecules that have specific affinity toward certain cellular components.

7. Specific interaction between molecules
If you are specifically interested in B, then ……

8. Tag with a label (e.g. fluorescence)!
Use to identify B and determine its (B’s) location in cells and tissues

9. Procedure Fixation (unless live cells are to be studied)
Permeabilization
Blocking
Labeling
Mounting

10. Procedure Fixation (unless live cells are to be studied)
Permeabilization
To allow penetration of fluorescent compounds
Blocking
Blocking sites prone to nonspecific interactions
Labeling
Mounting

11. Permeabilization
allow penetration of probes to gain access to the subcellular structures of interest
Not required for the localization of a cell surface structure
With detergent or organic solvents
Triton X-100, NP-40
Methanol, acetone

12. Labeling Plasma membrane Cytoskeleton Nucleus Mitochondria
NBD-PE, Fluorescent lipid analog (e.g. DiI,…), FITC- WGA
Cytoskeleton
phalloidin
Nucleus
DAPI, Hoechst, acridine orange, ethidium bromide
Mitochondria
Rhodamine123, Mito Tracker

13. DiI : Fluorescent lipid analog
Phospholipid

14. Fluorescent dye
(e.g. FITC) (e.g. RITC)

15. Sugar of the plasma membrane
FITC- WGA
fluorescein isothiocyanate
Wheat Germ Agglutinin
WGA is a plant lectin.
Different Lectins binds to specific sugars (glycoproteins, proteoglycans, glycoipids of the membrane).
Fluorescently labeled lectins
WGA (lectin)
Sugar of the plasma membrane
FITC
(fluorescence)

16. DAPI
DNA
phalloidin
F-actin
F-actin
phalloidin
FITC

17. DAPI phalloidin DNA Rhodamine (RITC) 4’,6-diamino-2-phenylindole
F-actin
phalloidin
Rhodamine
(RITC)

18. Acridine orange
DNA & RNA

19. Hoechst Mito Tracker DNA (nucleus) Mitochondria Labeling Live Cells!
(tetramethylrhodamine)
Mitochondria
Membrane-potential-sensitive
Labeling Live Cells!

20. BODIPY FL C5-ceramide Hoechst Golgi apparatus DNA (nucleus)
bovine pulmonary artery endothelial cell

21. Collage of images of cyanobacteria

22. Learning Resources Junqueira’s Basic Histology ; pp 5 & 12
Looking at the Structure of Cells in the Microscope
“Specific Molecules Can Be Located in Cells by Fluorescence Microscopy”

23. LESSON11 Detection Methods for specific proteins and genes
Immunohistochemistry
In situ hybridization
Green fluorescent protein (GFP)

24. Immunohistochemistry (IHC)
method of detecting the presence of specific proteins(/molecules) in cells (or tissues) using modified (labeled) antibodies .

25. Antibody جسم مضاد
react specifically and bind to the antigen
For IHC we must have an antibody against the protein to be detected (protein of interest).

26. So these Antibodies will ‘stick’ to the antigens in our fixed cells
So these Antibodies will ‘stick’ to the antigens in our fixed cells. But how do we see them?

27. Of course we’re going to add a label (tag).
Fluorescent compounds
Enzyme (peroxidase, alkaline phosphatase……)
Electron-dense gold particles

28. Direct method
Antibody against the protein of interest is tagged itself directly with a label

29. Amplification of Signals!
Indirect method
Primary Antibody specific for protein X.
Secondary Antibody tagged with a label ,
against the immunoglobulin class to which the primary antibody belongs.
(e.g. rabbit anti-mouse antibodies)
Amplification of Signals!

30. Primary Antibody against Desmin (Intermediate filament)
FITC-labeled Secondary Antibody
DAPI

31. Primary Antibody against lysozyme
Secondary Antibody labeled with peroxidase
Hematoxylin

32. (e.g. DAB)
(e.g. peroxidase)

33. Antibody against the amylase Protein A coupled with gold particles.
Protein A has high affinity toward antibody molecules.

34. The Avidin–Biotin Complex (ABC) Method

35. Information flow
Can we detect specific sequence of DNA or RNA?

36. In situ hybridization (ISH)
method of detecting the presence of specific DNA or RNA sequence in cells (or tissues) using labeled complementary DNA or RNA strand (probe)

37. 1. Determining the localization of a gene in a specific chromosome

38. 1. Determining the localization of a gene in a specific chromosome

40. 2. Identifying the cells containing specific mRNAs

41. Whole mount in situ hybridization

43. Green fluorescent protein (GFP) بروتينات فلورية خضراء
“A green guiding star for biosciences”
to track protein of interest in cells (or tissues)
“Color your protein!”

46. Tumor surrounded by nourishing blood vessels

47. Living neruon
GFP-mice

48. Learning Resources Junqueira’s Basic Histology ; pp 12 - 15
Looking at the Structure of Cells in the Microscope