Lesson 10 Fluorescence Staining =Fluorescent labeling

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

Why fluorescence? Contrast! Brilliant signals against dark background

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

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

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.

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

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

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

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

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

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

DiI : Fluorescent lipid analog Phospholipid

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

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)

DAPI DNA phalloidin F-actin F-actin phalloidin FITC

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

Acridine orange DNA & RNA

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

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

Collage of images of cyanobacteria

Learning Resources Junqueira’s Basic Histology ; pp 5 & 12 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK28356/ “Specific Molecules Can Be Located in Cells by Fluorescence Microscopy”

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

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

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).

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?

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

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

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!

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

Primary Antibody against lysozyme Secondary Antibody labeled with peroxidase Hematoxylin

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

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

The Avidin–Biotin Complex (ABC) Method

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

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)

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

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

2. Identifying the cells containing specific mRNAs

Whole mount in situ hybridization

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!”

Tumor surrounded by nourishing blood vessels

Living neruon GFP-mice

Learning Resources Junqueira’s Basic Histology ; pp 12 - 15 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK28356/