獸獸獸獸獸獸獸獸 獸獸獸 - - 獸獸獸
獸醫基礎科學概論獸醫基礎科學概論
- 形態學 -
陳建榮
The role of morphological studies in researchThe role of morphological studies in research
研究主題研究主題 Neuronal plasticity神經細胞的可塑性
Sex hormones E2 雌激素Testosterone 睪固酮
Hepatic encephalopathy 肝腦症 Central fatigue 中樞疲勞 Intracerebral hemorrhage 顱內出血
Physical compression 物理性壓迫Chemical stimulation 化學性刺激
3D reconstruction of tissue Urethra (female rat)
常用的研究方法常用的研究方法 Morphological methods
Histological staining 組織化學染色 IHC 免疫組織化學染色 Neuronal tracing 神經順向或逆向追蹤 Intracellular dye injection 細胞內染料注射
Electrophysiological methods Intracellular recording (current clamp) 細胞內電生理
Behavioral methods Rota-rod test 滾輪測試 Weight-loaded forced swimming test 負重游泳測試 Activities test 活動力測試 Morris water maze 水迷宮
Rota-rod test
Weight-loaded forced swimming test
Morris water maze
TopicsTopics
Stem cells 幹細胞 Intracellular electrophysiological
recording 細胞內電生理 Patch clamp Current clamp Field potential recording
Immunohistochemical (IHC) staining 免疫組織化學染色 Culture IHC-F, IHC-P WB
7
Section ISection I
The application of stem cells
RegenerationRegeneration
P. Anversa, NEJM, 2002
L. Iten, 1973
K. Poss, Science, 2002
Stem cellsStem cells
Features Self-renewal 自我更新 Potency 分化潛能
Sources Embryonic stem cells Adult stem cells
Classification Totipotent stem cells 全能幹細胞 Pluripotent stem cells 多功能幹細胞 Multipotent stem cells 多潛能幹細胞 Unipotent stem cells 專一性幹細胞
The sources of Pluripotent stem cellsThe sources of Pluripotent stem cells Inner cell mass
http://php.med.unsw.edu.au/embryology/index.php?
Bone marrow 骨髓 Umbilical cord 臍帶 Adipose tissue 脂肪組織 Endothelial cell 內皮細胞 Dental pulp 牙髓 Amniotic fluid 羊水 Induced pluripotent stem cells (iPS) 誘導多功能幹細胞
The sources of stem cells
Embryonic stem cell Adult stem cell
Type Embryonic stem cells are
pluripotent
Adult stem cells are limited to
differentiating
Culture Embryonic stem cells grown easily
in culture
Adult stem cells are rare in mature
tissues
Transplant rejection
Embryonic don't yet know
Adult stem cells, less likely
to initiate rejection
The Applications of Stem cellsThe Applications of Stem cells
細胞、組織、器官修補更新腦中風、中樞神經退化心衰竭…
人造器官與組織的來源 新藥開發 基因功能研究 基因治療的工具 毒理、藥理研究 癌症研究
Stem Cell TherapyStem Cell Therapy
M. Mimeault, Stem Cell, 2006
Target disorder: myocardial infarction
M. Schneider, Nature, 2004 A. Mathur, Lancet, 2004
Stem Cells in Cardiac DisordersThe spotlight of regenerative medicine
The Applications of Stem cellsThe Applications of Stem cells
細胞、組織、器官修補更新 人造器官與組織的來源
外耳心臟
新藥開發 基因功能研究 基因治療的工具 毒理、藥理研究 癌症研究
人造心臟人造心臟
19
Section IISection II
Electrophysiological properties of the neurons
20
Basic Concepts
VoltA charge difference between two points
in spaceIons – charged particles
Anions – Negatively charged particlesCl-
Cations – Positively charged particlesNa+, Ca 2+, K+
21
Basic ConceptsForces that determine ionic movement
Electrostatic forces靜電的力量同性相斥,異性相吸
Concentration forces濃度的力量Diffusion 擴散Osmosis 反滲透
22
Calculating equilibrium potential
Nernst Equation
Allows theoretical membrane potential to be calculated for particular ion.Membrane potential that would exactly balance
the diffusion gradient and prevent the net movement of a particular ion.
Value depends on the ratio of [ion] on the 2 sides of the membrane.
23
Nernst equation能斯特方程式
Equilibrium potential 平衡電位 (mV) , Eion = lnRTzF
[C]o
[C]i
[C]o and [C]i = extra and intracellular [ion] R = Universal gas constant 氣體常數 (8.3 joules.K-1.mol-1)T = Absolute temperature 絕對溫度 (°K)F = Faraday constant 法拉第常數
是每莫耳電子所攜帶的電荷量 (96,500 coulombs.mol-1)z = Charge of ion 離子電荷 (Na+ = +1, Ca2+ = +2, Cl- = -1)
For K+, with [K+]o = 4 mmol.l-1 and [K+]i = 150 mmol.l-1
At 37°C, EK = -97mV, ENa = +60mV, ECl = -67mV
24
Selective Permeability of Membranes
Some ions permitted to cross more easily than others
Neuronal membranes contain ion channelsProtein tubes that span the membraneSome stay open all the time (nongated)Some open on the occasion of an action potential,
causing a change in the permeability of the membrane (gated)
25
Resting Membrane Potential靜止膜電位
Na+ and Cl- are more concentrated outside the cell
K+ and organic anions (organic acids and proteins) are more concentrated inside.
26
The Sodium-Potassium Pump
extrudes Na+ from the cell while taking in K +
27
The formation of resting potentialThe formation of resting potential
Concentration difference of K+ across the membrane
Permeability of Na+ and K+ during the resting state
Na+-K+ pump
Resting membrane potential (RMP)
28
Intracellular vs extracellular ion concentrations
Ion Intracellular Extracellular
Na+ 5-15 mM 145 mMK+ 140 mM 5 mMMg2+ 0.5 mM 1-2 mMCa2+ 10-7 mM 1-2 mMH+ 10-7.2 M (pH 7.2) 10-7.4 M (pH 7.4)
Cl- 5-15 mM 110 mM
29
Resting Membrane Potential Goldman (Goldman-Hodgkin-Katz) equation
E = 61.5 x log{(PK[K]o + PNa[Na]o + PCl[Cl]o) / (PK[K]i+PNa[Na]i+PCl[Cl]i)} ~ - 70 mV
Resting membrane potential of most cells ranges from - 65 to – 85 mV.
30
Basic Electrophysiological TermsBasic Electrophysiological Terms
Polarization極化 : a state in which membrane is polarized at rest, negative inside and positive outside.
Depolarization去極化 : the membrane potential becomes less negative than the resting potential (close to zero).
Hyperpolarization再極化 : the membrane potential is more negative than the resting level.
31
Action Potential動作電位
Successive Stages:
(1) Resting Stage
(2) Depolarization stage
(3) Repolarization stage
(4) After-potential stage
(1)
(2) (3)
(4)
32
Ion Permeability during the APIon Permeability during the AP
Figure 8-12: Refractory periods
33
Electrophysiological Method to Record Membrane Potential IElectrophysiological Method to Record Membrane Potential I
Voltage Clamp 電壓箝制Current Clamp 電流箝
制
34
The Nobel Prize in Physiology or Medicine (1963)
“for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane”
Alan Lloyd Hodgkin Andrew Fielding Huxley
35
The Hodgkin-Huxley Model of Action Potential Generation
36
Modern proof of
nature of currents
Use ion selective agents-河魨毒( TTX)-四乙胺 (TEA)
Intracellular electrophysiological recording
Membrane propertiesMembrane properties
Action potential
Current– voltage Relationships
V=I*R
Spike generating patterns
Electrical signals between neurons
Excitatory Post-Synaptic Potential Inhibitory Post-synaptic Potential
Reversal potential反轉動位
41
Electrophysiological Method to Record Membrane Potential IIElectrophysiological Method to Record Membrane Potential II
Patch Clamp 膜片箝制
42
"for their discoveries concerning the function of single ion channels in cells"
The Nobel Prize in Physiology or Medicine (1991)
Erwin Neher Bert Sakmann
Patch clamp recording
Inside out: The effects of intracellular molecular on receptor ion channel
outside out: The effects of extracellular molecular on receptor ion channel
45
How channel conductances accumulate
Whole-cell recordingWhole-cell recording
47
Electrophysiological Method to Record Membrane Potential IIIElectrophysiological Method to Record Membrane Potential III
Extracellular recording細胞外記錄
(Field potential)
Extracellular recording
Field Excitatory Post-synaptic Potential (fEPSP)
Synaptic potential allow transmission of information from one neuron to another
stimulate
EPSP LTP
induced by a short tetanus (10 pulses at 100 Hz) at hippocampal CA1 synapses
Procedures of electrophysiological studiesProcedures of electrophysiological studies
Decapitated Vibratome section Culture Recording
Vibratome section
Conservation of brain slice activity
Artificial cerebrospinal fluid人工腦脊髓液 (ACSF)NaCl, KCl, CaCl, MgCl2, NaHCO3 NaH2PO4, glucose
Energy Glucose + 95% Oxygen and 5% CO2
Slice preparation 4 in ACSF (without Ca℃ 2+)
Slice maintenance 25 in ACSF℃
Patch clamp assemblyPatch clamp assembly
Patch clamp assembly. Single‐channel currents are amplified, filtered, digitized, and stored in video tapes and/or computer discs. The components of patch clamp assembly required for tip‐dip bilayer technique are shown: (1a) microbeaker; (1b) artificial extracellular fluid; (1c) microstir bar; (1d) lipid monolayer; (1e) lipid bilayer; (1f) patch pipette; (1g) artificial intracellular fluid; (1h) recording electrode; (1i) reference electrode; (1j) electrode holder; (1k) head stage; (2) head stage; (3) Faraday box; (4) isolation table; (5) patch clamp amplifier; (6) video cassette recorder; (7) analog‐to‐digital converter; (8) low‐pass filter; (9) digitizer; (10) oscilloscope; (11) computer hard drive; (12) monitor; (13) printer.
http://www.sciencedirect.com/science/article/pii/S007668790617007X
amplifier
analog‐to‐digital converter
digitizer
Current clamp
Copper mesh銅網
Micropipette Puller
Extracellular recordings with Microelectrode arrays微電極陣列
Extracellular recordings with Microelectrode arrays微電極陣列
57
Section IIISection IIIImmunohistochemical
(IHC) stainingAntibody
Polyclonal antibody 多株抗體 Monoclonal antibody 單株抗體
IHC Staining ProtocolIHC Staining Protocol
Fixation 4% paraformaldehyde Transcardial perfusion antigen retrieval protocols
Section Paraffin section Cryosection
Antigen - Antibody reaction (Temperature)
Enzymes and Chromogens
Transcardial PerfusionTranscardial Perfusion
Antigen Retrieval抗原還原Antigen Retrieval抗原還原
During the process of formalin fixation, many antigenic sites are ‘masked’ and are therefore sometimes difficult or impossible to stain without antigen retrieval.
Antigen retrieval is a process of treating formalin fixed-paraffin embedded tissue sections with proteolytic enzymes or heating them in various buffer solutions in order to expose the antigen.
Commonly used proteolytic enzymes include trypsin, pepsin and protease.
56
Antigen RetrievalAntigen Retrieval
Heat induced epitope retrieval (HIER) includes microwaving, pressure cooking, steaming, autoclaving or using the PreTreatment Module™.
Requires buffer of different concentrations and pH. Commonly used buffers include
Citrate buffter at pH 6.0 EDTA at pH 8.0 Tris-HCL at pH 10.0
57
Antigen RetrievalAntigen Retrieval
No Antigen RetrievalCitrate buffer, pH 6.0
58
These photos show the staining results of CD3 antibody on tonsil, with and without antigen retrieval.
Paraffin sectionParaffin section
CryosectionCryosection
IHC Staining MethodsIHC Staining Methods
69
Direct Method Indirect Method
Two-Step Three-Step
Avidin-Biotin Complex (ABC) Method
Direct MethodDirect Method
It has the advantages of rapidity, ease of performance and limited nonspecific reaction.
73
Enzymeor fluorescentprimary Ab
antigen
Direct IHC Staining Direct IHC Staining
E-Cadherin and DAPI
Adiponectin in Mouse skin
Indirect MethodIndirect Method Uses an enzyme-labeled secondary antibody that is
directed against the unlabeled primary antibody.
If the primary antibody (which is now the antigen) is made in mouse, the secondary antibody must be against mouse immunoglobulin.
More sensitive than the Direct Method because several secondary antibodies are likely to bind with a number of various epitopes on the primary antibody increasing the enzyme labels involved.
76
Indirect Method - ProcedureIndirect Method - Procedure
An unlabeled primary antibody binds to the tissue antigen.
77
primary Ab (mouse)
antigen
Two-Step Indirect MethodTwo-Step Indirect Method
An enzyme-labeled secondary antibody binds to the primary antibody.
78
secondary Ab(rabbit anti-mouse)
Enzyme or fluorescent
Indirect IHC Staining Indirect IHC Staining
CD133 antibody (Stem Cell Marker) and DAPI
Multiple ImmunolabelingMultiple Immunolabeling
Multiple immunofluorescence labeling of fixed sections of breast cancerA formalin-fixed paraffin-embedded section of a breast cancer positive for estrogen receptor (ER). An area of ductal carcinoma shows high-resolution four-color immunolabeling after in situ staining for cytokeratin 8/18 (green), ER (red) and vimentin (yellow). Nuclei were counterstained with DAPI (blue). http://www.abcam.com/
Rat cortical neurons and glia in mixed tissue culture stained with MAP2 (green) and GFAP (red). Nuclei of all cells are stained with Hoechst dye (blue).
http://www.abcam.com/
Small intestine stained with anti-CD10 and DAB+Ni substrate (gray/black). Cytokeratin 20 is visualized with ImmPRESS™ Anti-Mouse Ig and Vector® NovaRED™ substrate (red)
http://www.abcam.com/
Three-Step Indirect MethodThree-Step Indirect Method
An enzyme-labeled tertiary antibody is added and binds to the secondary antibody.
85
tertiary Ab(goat anti-rabbit)
Avidin-Biotin MethodsAvidin-Biotin Methods Uses the strong and high affinity of avidin (egg white
glycoprotein) for biotin (water-soluble vitamin).
Avidin has four binding sites for biotin but fewer than four molecules of biotin will actually bind to avidin.
100
avidinbiotin
molecules
Avidin-Biotin MethodsAvidin-Biotin Methods
Two of the most common methods include Avidin-Biotin enzyme Complex (ABC) Labeled StreptAvidin-Biotin (LSAB)
The inherent amplification of sensitivity offered by avidin and biotin makes these methods more favorable than PAP or APAAP.
Streptavidin, a bacterial protein has recently replaced avidin because it produces less background staining than avidin.
101
The enzyme complex is prepared by mixing biotinylated enzyme (HRP or AP) and avidin.
ABC MethodABC Method
avidin-biotin-enzyme complex
103
This preformed avidin-biotin-enzyme complex then reacts with the biotinylated secondary antibody.
avidinbiotinylatedenzyme
ABC - ProcedureABC - Procedure
An unlabeled primary antibody binds to the antigen.
104
antigen primary Ab(mouse)
ABC - ProcedureABC - Procedure
A biotinylated secondary antibody binds to the primary antibody.
105
secondary Ab(rabbit anti-mouse)
biotin
ABC - ProcedureABC - Procedure
A preformed avidin-biotin-enzyme complex solution is added and binds to the biotinylated secondary antibody.
106
avidin-biotin-enzyme complex
ABC - ProcedureABC - Procedure
A substrate-chromogen solution is added ending the reaction and producing a colored end-product.
107
substrate-chromogen
substrate-chromogen
LSAB MethodLSAB Method
Uses enzyme-conjugated streptavidin. Streptavidin is conjugated to several molecules of enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP).
The secondary antibody is conjugated to numerous biotin molecules, each of which can potentially bind to an enzyme-conjugated streptavidin.
109
LSAB – ProcedureLSAB – Procedure
An unlabeled primary antibody binds to tissue antigen.
110
antigen
primary Ab
LSAB – ProcedureLSAB – Procedure
A biotinylated secondary antibody binds to the primary antibody.
Each secondary antibody contains multiple biotin molecules; several secondary antibodies can bind to the primary antibody.
111
biotin
secondary Ab
LSAB – ProcedureLSAB – Procedure
An enzyme-labeled streptavidin is added and binds to the secondary antibody.
HRP-streptavidin
112
LSAB – ProcedureLSAB – Procedure
A substrate-chromogen solution is added producing a colored end-product.substrate-
chromogen
113
Enzymes and ChromogensEnzymes and Chromogens
Detection systems attach enzyme labels to primary or secondary antibodies to visualize the localized antibody-antigen binding in tissue section.
Enzymes are proteins that act as catalysts to increase the rate of chemical reaction. They are used in IHC to convert a colorless reagent into a stable colored product (chromogen) that marks the site of antibody-antigen complex.
A chromogen is a substance that absorbs light, producing color.
60
Enzymes and ChromogensEnzymes and Chromogens
Commonly used enzyme labels for IHC procedures include
horseradish peroxidase (HRP) 過氧化物酶 alkaline phosphatase (AP) 鹼性磷酸酶
HRP is an enzyme that catalyze the reduction of hydrogen peroxide (H2O2) to water and oxygen.
Commonly used chromogens for HRP include
3-amino-9-ethylcarbazole (AEC) 3,3’-diaminobenzidine (DAB)
61
AEC is oxidized by HRP producing a bright red reaction product. This reaction product is not stable and may fade over time.
AECAEC
62
C2H5
N
Structure of AEC
NH2
DAB is oxidized by HRP producing a dark brown reaction product. This reaction product is stable and does not fade over time.
DABDAB
64
Structure of DAB
NH3+
NH3+
+H3N
+H3N4Cl
-
AEC and DABAEC and DAB
AEC chromogenMart-1
Melanoma
DAB chromogenMart-1
Melanoma
66
Examples of staining results using AEC and DAB chromogens.
Stained Pattern - Cell MembraneStained Pattern - Cell Membrane
c-erbB-2Breast
carcinomaAEC chromogen
CD3/T-CellTonsil
AEC chromogen
136
Stained Pattern - NuclearStained Pattern - Nuclear
Estrogen Receptor (ER)
Breast carcinomaAEC chromogen
Cyclin D1Mantle cell lymphoma
DAB chromogen
137
Stained Pattern - CytoplasmicStained Pattern - Cytoplasmic
Keratin, PanColon carcinomaAEC chromogen
VimentinMelanoma
AEC chromogen
138
Stem cellsStem cells
Features Self-renewal 自我更新 Potency 分化潛能
Sources Embryonic stem cells Adult stem cells
Classification Totipotent stem cells 全能幹細胞 Pluripotent stem cells 多功能幹細胞 Multipotent stem cells 多潛能幹細胞 Unipotent stem cells 專一性幹細胞
Gurdon JB Shinya Yamanaka( 山中伸彌 )
Induced pluripotent stem cells (iPS) 誘導多功能幹細胞
The Nobel Prize in physiology or medicine 2012The Nobel Prize in physiology or medicine 2012
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Resting Membrane Potential Goldman (Goldman-Hodgkin-Katz) equation
E = 61.5 x log{(PK[K]o + PNa[Na]o + PCl[Cl]o) / (PK[K]i+PNa[Na]i+PCl[Cl]i)} ~ - 70 mV
Resting membrane potential of most cells ranges from - 65 to – 85 mV.
Inside out recording: The effects of intracellular molecular on receptor ion channel
outside out recording: The effects of extracellular molecular on receptor ion channel
Conservation of brain slice activity
Artificial cerebrospinal fluid人工腦脊髓液 (ACSF)NaCl, KCl, CaCl, MgCl2, NaHCO3 NaH2PO4, glucose
Energy Glucose + 95% Oxygen and 5% CO2
Slice preparation 4 in ACSF (without Ca℃ 2+)
Slice maintenance 25 in ACSF℃
IHC Staining ProtocolIHC Staining Protocol
Fixation 4% paraformaldehyde Transcardial perfusion antigen retrieval protocols
Section Paraffin section Cryosection
Antigen - Antibody reaction (Temperature)
Enzymes and Chromogens
Direct MethodDirect Method
It has the advantages of rapidity, ease of performance and limited nonspecific reaction.
73
Enzymeor fluorescentprimary Ab
antigen
Avidin-Biotin MethodsAvidin-Biotin Methods
Two of the most common methods include Avidin-Biotin enzyme Complex (ABC) Labeled StreptAvidin-Biotin (LSAB)
The inherent amplification of sensitivity offered by avidin and biotin makes these methods more favorable than PAP or APAAP.
Streptavidin, a bacterial protein has recently replaced avidin because it produces less background staining than avidin.
101
Enzymes and ChromogensEnzymes and Chromogens
Commonly used enzyme labels for IHC procedures include
horseradish peroxidase (HRP) 過氧化物酶 alkaline phosphatase (AP) 鹼性磷酸酶
HRP is an enzyme that catalyze the reduction of hydrogen peroxide (H2O2) to water and oxygen.
Commonly used chromogens for HRP include
3-amino-9-ethylcarbazole (AEC) 3,3’-diaminobenzidine (DAB)
61
Polyclonal Antibody ProductionPolyclonal Antibody Production
25
A rabbit is injected subcutaneously with a purified dose of antigen.
The rabbit’s immune system responds by producing antibodies specific to the injected antigen.
Blood is harvested from the ear at the peak of antibody production.
Red blood cells and clotting proteins are removed and the antiserum is purified.
YYY
YYY
Polyclonal AntibodiesPolyclonal Antibodies
22
Polyclonal antibodies reacting with various epitopesEach antibody is made by a different B-cell
Polyclonal Antibody ProductionPolyclonal Antibody Production Polyclonal antibodies are purified either by Protein
Purification or Antigen Affinity Chromatography.
Protein Purification eliminates the bulk of serum proteins but does not eliminate nonspecific immunoglobulin fraction.
Antigen Affinity Purification eliminates the bulk of the nonspecific immunoglobulin fraction using antigen to capture the antibody leaving only the immunoglobulin of desired specificity.
28
Monoclonal AntibodiesMonoclonal Antibodies Monoclonal antibodies are derived from a single B-cell
and are produced as a single class of immunoglobulin.
They are raised by fusion of the specific B-cells with immortal myeloma (B-cell) cancer cells to form a hybridoma.
A hybridoma can multiply indefinitely and continuously produce a specific monoclonal antibody.
They react with a specific epitope on a given antigen, giving less background staining.
30
Monoclonal Antibody ProductionMonoclonal Antibody Production
34
A mouse is injected subcutaneously with a purified dose of antigen.
The mouse’s immune system responds by producing antibodies specific to the injected antigen.
The antibody-producing B-cells are harvested from the spleen or lymph nodes.
Monoclonal Antibody ProductionMonoclonal Antibody Production
B-lymphocytes
spleen
36
The B-cells are fused with mouse myeloma cells forming immortal hybrid cells or hybridomas.
Monoclonal Antibody ProductionMonoclonal Antibody Production
myeloma cells
37
B-lymphocytes
The generated hybridomas will produce many copies of the exact same antibody.
Monoclonal Antibody ProductionMonoclonal Antibody Production
38
Monoclonal Antibody ProductionMonoclonal Antibody Production
39
The hybridomas:Transplanted into the peritoneal cavityPropagated in a tissue culture medium
Monoclonal AntibodiesMonoclonal Antibodies
Monoclonal antibodies reacting with similar epitopes
32
Antibody Titer and DilutionAntibody Titer and Dilution
1:8001:400
1:50 1:100 1:200
47
Incubation TimeIncubation Time
Incubation time is inversely proportional to antibody concentration. Higher concentration of antibody allows shorter incubation time.
It can be from minutes to hours, with 30-60 minutes the most common practice.
52
Incubation TemperatureIncubation Temperature Antibody-antigen reaction is hastened at
37C as compared to room temperature. An increase in temperature also allows for a higher dilution of the antibody.
Humidity chambers must be used when incubating at higher temperature to prevent drying of tissue sections.
54