Teaching)

The Technique for Morphology Visualizing cells Lan Bao Institute of Biochemistry and Cell Biology Chinese Academy of Sciences [email protected]

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

A sense of scale between living cells and atoms

From Molecular Biology of the Cell, 4th edtion

A light microscope

From Molecular Biology of the Cell, 4th edtion

Two ways to obtain contrast in light microscopy

A stained tissue section From Molecular Biology of the Cell, 4th edtion

Four types of light microscopy

(A) Bright-field microscopy. (B) Phase-contrast microscopy. (C) Nomarski differential-interference-contrast microscopy. (D) Dark-field microscopy From Molecular Biology of the Cell, 4th edtion

An inverted microscope

Stereomicroscope

A fluorescence microscope

From Molecular Biology of the Cell, 4th edtion

Fluorescent dyes

From Molecular Biology of the Cell, 4th edtion

A confocal microscope

From Molecular Biology of the Cell, 4th edtion

Conventional and confocal fluorescence microscopy

From Molecular Biology of the Cell, 4th edtion

Transmission electron microscope

From Molecular Biology of the Cell, 4th edtion

Shematic drawing of an idealized cell with organelles

Scanning electron microscope

From Molecular Biology of the Cell, 4th edtion

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

Routine staining of tissue sections Hematoxylin-and-eosin, or H&E staining, is used for routine staining of tissue sections. Hematoxylin, a basic dye, binds to acidic components of a tissue, which are thus said to be "basophilic." The color of the stained structures depends on the mordant used to make the hematoxylin dye bind to the molecules of the tissue. Potassium alum, the most common mordant, gives the dye a blue to purple color. Basophilic nuclei, bacteria, calcium, and so on are stained "blue" with hematoxylin. Eosin, an acidic dye, binds to basic components of a tissue, which are thus said to be "acidophilic." The structures stained by eosin are typically colored pink to red. Eosinophilic cytoplasm, connective, and all other tissues are counterstained "red" with eosin.

H&E-staining of mouse salivary glands fixed in formalin

Structures made visible by special stains Other cellular structures can only be identified in the light microscope if special stains are used. Cresyl Violet (Nissl stain) Nissl stain stains RNA and DNA purple. The socalled Nissl substance in the cytoplasm of nerve cells has been identified as ribosomes and the rough endoplasmic reticulum. This stain gives the Nissl substance a purple color.

This section from the spinal cord has been stained with cresyl violet, known as the Nissl stain, which stains RNA.

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

Definition: The use of labeled antibodies as specific reagents for localization of tissue/cell constituents (antigens) in situ.

Antigen-antibody reaction Methods: Direct/Indirect Immunofluorescence single-, double-, triple- labeling Immunoperoxidase

Simplified diagram of IgG molecule

Immunofluorescence labeling Direct immunostaining

Indirect immunostaining

Immunofluorescence labeling single immunostaining

double immunostaining

Dorsal root ganglion neuron

Y1/CGRP

DOR/SP

HEK293 cell

2 weeks after nerve injury

Con

Tong, et al. JCN 1999

Delta- and Mu-opoid receptor

Delta

Mu

Zhang X, Bao L, et al (1998a, b) Neuroscience

Immunoperoxidase labeling

Avidin-Biotin-Peroxidase

From Vector Laboratory

Immunoperoxidase micrographs of cholera toxin B-subunit conjugated to horseradish peroxidase (CTB-HRP) labelling in ipsilateral (Ipsi) and contralateral (Con) rat dorsal root ganglia 18 days after unilateral sciatic nerve transection. Tong, et al. JCN 1999

Essential conditions for immunocytochemistry: 2. Preservation of the antigen in tissue context (fixation) 3. Well characterized antibodies 3. Specific and sensitive staining with absence of non-specific staining 4. Efficient detection

Fixation: Fixative （ 4% paraformaldehyde, picric acid ） Cross-linking (paraformaldehyde, glutaraldehyde) Precipitant fixatives (alcohol, methanol, acetone) pH （ acidic fixative, neutral fixative ） Procedure （ perfusion, immersion, post-fixation ） Sucrose （ 10% 、 20% 、 30% ）

Preparation and storage of specimens: 2. Frozen sections on adhesive-coated glass slides 3. Cell smears and cytocentrifuged cells 4. Fixative-fixed, paraffin-embedded sections on adhesive-coated glass slides.

Primary antibody: Host （ Mo, Rb, Go, GP, Ch ） Application （ IB, IH, IP, IC) Cross reaction （ Rt, Mo, Hu, Rb) Storage (4℃, -20℃, -70℃ ) Characteristics of a ‘good’ antibody: high affinity, high avidity (stickiness), high titre or concentration

Secondary antibody: Specificity （ Mo, Rb, Go, GP, Ch ） Fluorescence （ FITC, Rhodamine, Cy3, Cy5 ) Specificity （ minx Rt, Mo, Hu)

Immunohistochemistry Controls Positive Staining Control Antibody Positive Control: Stain tissues known to contain the antigen under investigation Negative Staining Controls Null Control: Omit primary antibody and incubate tissues with buffer Absorption: Incubate antigen with antibody for 24 hours, and then incubate the tissue with this buffer

Problems and remedies High level of background staining 1. Dilute the primary antibody further. 2. Check negative control with second primary antibody. Absorption with tissue powder (acetone-dried liver) 3. If background is still present, it could be due to: (f) Tissue factors (non—specific binding sites, Fc receptors, basic proteins) Remedy: (i) Increase concentration of blocking protein/serum. (ii) Add detergent to buffer rinse (0.05% Tween 20). (iii) Raise NaCl content of antibody diluent to 2.5%. (iv) Raise pH of buffer to 9.0. (v) Use F(ab) portions of antibodies. (vi) Add 2 mg poly-L-lysine (a basic protein to each ml of diluted antibody. (n) Cross-reaction between anti-species immunoglobulin and host-tissue immunoglobulins Remedy: (i) Absorb cross-reacting antibody with 1% of host-tissue species normal serum or immunoglobulin. (ii) Use species-specific antibodies.

Problems and remedies (c) Aldehyde groups in tissue left from fixative Remedy: (I) Wash tissue well before processing and embedding. (ii) Treat preparation with freshly made 0.02-1% sodium or potassium borohydride in 0.1 M phosphate buffer or water for 2-30 min at RT. (iii) Add 10-100 mM NH4Cl to the blocking serum. Immunostaining weak or absent 9. Method sensitivity is insufficient for small quantity of antigen present Remedy: Increase sensitivity. 11. Antigen is hidden (over-fixed). Remedy: (i) Antigen retrieval by protease treatment (ii) Heat-mediated antigen retrieval 3. Antibody deterioration 4. Wrong antibody sequence applied in error

Blocking of unwanted non-specific staining: Source of unwanted staining, besides poor knowledge of the antibody reactivity and malice, is due to: •Endogenous enzymes or fluorochromes. •Endogenous biotin. •Endogenous antibody binding activity (Fc receptors). •Crossreactivity of the secondary reagents with endogenous proteins.

Blocking of endogenous enzymes Endogenous enzymes such as AlkPhos, AcPhos and esterases are destroyed by boiling, even a short time at 100 C. Peroxidase is not. Blocking of endogenous peroxidase is done by preincubating the slides in 3% H2O2 . Blocking of endogenous fluorochromes. Blocking of endogenous fluorochromes is impossible. One may choose fluorochromes emitting in the UV range of spectrum, where endogenous autofluorescence of tissue is minimal.

Blocking endogenous biotin Can be done with commercially available kits or by buying the isolated components of the kits, free biotin and free avidin. Blocking of endogenous Fc blocking. Specimens not paraffin embeddded may have significant Fc binding activity by macrophages, B cells, T cells and other cell types. By exploiting the preferential avidity of Fc receptor for human > mouse Ig> rabbit > swine > goat immunoglubulins, one may use a blocking of the receptors with a reagent which will not interfere with the secondary reagents and with which the secondary antibodies can be absorbed (1% serum added).

Blocking of crossreactive antigens in the tissue. Typical example is staining mouse monoclonals in mouse tissue, where endogenous immunoglobulins will be specifically detected by the antibody aimed at the exogenous antibody used.

Three-dimensional reconstruction from confocal microscope images

Three-dimensional reconstruction from confocal microscope images Neuropeptide Y/ CTB

Three-dimensional reconstruction from confocal microscope images

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

Routine Processing For Electron Microscopy • •

• • •

Tissue is fixed in EM fixative (2.5% glutaraldehyde or 4% paraformaldehyde/1% glutaraldehyde). The tissue is post-fixed in 1% osmium tetroxide(OsO4) to preserve lipid and lipoprotein structures. The specimen is then processed through a graded series of alcohols, infiltrated in Spurr epoxy resin, embedded and polymerized. An appropriate area are selected for ultra-thin sectioning with a diamond knife. Sections are placed on grids, stained with uranyl acetate and lead citrate, and examined by EM and photographed.

Nuclear pore

Nucleus

Rough ER

Golgi apparatus

Smooth ER

Secretary granule

Endosome

Multivesicular body

Secondary lysosome

Primary lysosome

Tertiary lysosome

Myelinated fibre

Synapse

Ranvier node

Mitochondria

Immunoelectron Microscopy This technique allows the investigator to identify antibody/antigen complexes that localize to a particular subcellular organelle or compartment.

Pre-embedding immunoelectron microscopy Post-embedding immunoelectron microscopy

e-embedding immunoelectron microscop • • • •

• •

Tissue is fixed in weak EM fixative (4% paraformaldehyde/0.05% glutaraldehyde). An antigen-antibody reaction can be done on Vibratome section including the tissue and cultured cell. The tissue is post-fixed in 1% osmium tetroxide(OsO4) to preserve lipid and lipoprotein structures. The specimen is then processed through a graded series of alcohols, infiltrated in Spurr epoxy resin, embedded and polymerized. An appropriate area is selected for ultra-thin sectioning with a diamond knife. Sections are placed on grids, stained with uranyl acetate and lead citrate, and examined by EM and photographed.

e-embedding immunoelectron microscop Pre-embedding immunoperoxidase technique Advantage: Good for immunoreactivity Disadvantage: 1. The antigen can not be precisely locate at subcellular level, especially the cytoplasm membrane and vesicle. 2. The subcelluler structure can not be well preserved. 3. DAB is carcinogen and should be careful for manipulations.

Neuropeptide Y Y1 receptor in arterioles

Bao, et al. PNAS 1997

e-embedding immunoelectron microscop Pre-embedding immunogold-silver labeling technique Advantage: 1. Good for immunoreactivity. 2. The antigen can be precisely locate at subcellular level. 3. Double labeling can be used with preembedding immunoperoxidase technique. Disadvantage: 1. The labeling is destroyed with osmium tetroxide (OsO4) post-fixation. 2. The subcelluler structure can not be well preserved.

Delta-opoid receptor

Zhang, et al. Neuroscience 1998

st-embedding immunoelectron microsco • • •

• • •

Tissue is fixed in weak EM fixative (4% paraformaldehyde/0.05% glutaraldehyde). The tissue is post-fixed in 1% osmium tetroxide (OsO4) to preserve lipid and lipoprotein structures. The specimen is then processed through a graded series of alcohols, infiltrated in Spurr epoxy resin, embedded and polymerized. An appropriate area is selected for ultra-thin sectioning with a diamond knife. Sections are placed on grids. An antigen-antibody reaction can be done on ultra-thin section. The sections are stained with uranyl acetate and lead citrate, and examined by EM and photographed.

t-embedding immunoelectron microscop Post-embedding immunogold labeling Advantage: 1. The antigen can be precisely located at subcellular level. 2. Multi-labeling can be used. 3. The subcelluler structure can be well preserved. 4. It’s good for the labeling of the neurotransmittor of amino acid (GABA). Disadvantage: Bad for immunoreactivity.

Colocalization of Delta-opoid receptor with CGRP

DOR/CGRP Bao, et al. Neuron 2003

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immuno-electron microscopy Fluorescence dye Quantitative analysis Application

• • • • •

Nucleic acid detection Actin and tublin probes Apoptosis probes Calcium indicator Organelle probes: golgi apparatus, endoplasmic reticulum, lysosomes, vacuoles and other acidic organelles, mitochondria • Photoactivatable (caged) probes

Nucleic acid detection

Golgi bodies in a muntjac skin cell were labeled with anti–Golgin-97 antibody and visualized with green-fluorescent Alexa Fluor 488 goat anti–mouse IgG1 antibody. Filamentous actin was labeled with Alexa Fluor 680 phalloidin (pseudocolored purple). Nuclei were stained with blue-fluorescent DAPI. From Molecular Probes

Actin and tublin probes

Actin filaments of the turbellarian flatworm Archimonotresis sp. stained with Alexa Fluor 488 phalloidin to reveal a meshwork of longitudinal, circular and diagonal muscles. From Molecular Probes

Apoptosis probes

Detection of apoptosis in SK-N-MC neuroblastoma cells Hoechst 33342 (blue), Tetramethylrhodamine ethyl ester (TMRE, red), mitochondria marker Rhodamine 110, bis-L-aspartic acid amide (green), apoptosis marker From Molecular Probes

Calcium Indicators

REF-52 fibroblasts loaded with fura-2 AM following microinjection of Cyclic AMP Fluorosensor (FlCRhR) From Molecular Probes

MitoTracker

Bovine pulmonary artery endothelial cells incubated with MitoTracker Red CMXRos to label the mitochondria. After fixation and permeabilization, the cells were stained with BODIPY FL phallacidin to label the F-actin filaments and finally counterstained with DAPI to label the nucleus. From Molecular Probes

ER-Tracker

Live bovine pulmonary artery endothelial cells stained with ER-Tracker Blue-White DPX and MitoTracker Red CM-H2XRos. The endoplasmic reticulum appears green and the mitochondria appear orange. From Molecular Probes

LysoTracker Golgi marker

Viable Madin-Darby canine kidney (MDCK) cells sequentially stained with BODIPY FL C5-ceramide, LysoTracker Red DND-99 and Hoechst 33258. From Molecular Probes

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

Quantitative analysis Number Length Intensity

Bao, et al. Eur. J. Neurosci. 2002

DOR agonist induces [Ca2+]i elevation and DOR redistribution via store-operated Ca2+ channel

Bao, et al. Neuron 2003

Translocation of delta-opioid receptor after DOR selective agonist treatment

DRG neuron

Bao, et al. Neuron 2003

Activation of DOR induces CGRP release

Bao, et al. Neuron 2003

Immunostaining of inserted DORs Incubating cells in a mixture of antibody against N-terminal of DOR and 1 µM Delt

Bao, et al. Neuron 2003

Okuse, et al. Nature 2002

Contents • • • • • • •

Microscopy Histological staining Immunohistochemistry/Immunocytochemistry Immunoelectron microscopy Fluorescence dye Quantitative analysis Application

Immunostaining of inserted DORs Incubating cells in a mixture of antibody against N-terminal of DOR and 1 µM Delt

Bao, et al. Neuron 2003

Visualizing molecules in living cells

quinacrine labeled vesicles

Fura-2

Visualizing molecules in living cells

Visualizing molecules in living cells

Gómez-Moutón, et al. JCB 2004

Visualizing molecules in living cells

TrkB receptor Heerssen, et al. Nature Neurosci. 2004

Fluorescence Resonance Energy Transfer (FRET)

A-GFP

B-cy3

+C

Flourescence Recovery After Photobleaching (FRAP)

before photobleaching

recovery of fluorescence after photobleaching

after photobleaching

Graphical presentation of data collected during a FRAP experiment

Contents • Microscopy • Histological staining • Immunohistochemistry/Immunoc ytochemistry • Immunoelectron microscopy • Fluorescence dye • Quantitative analysis • Application