
f18274bf0cc737592808a19f268fc305.ppt
- Количество слайдов: 122
獸醫基礎科學概論 - 形態學 - 陳建榮
The role of morphological studies in research
研究主題 w Neuronal plasticity神經細胞的可塑性 w Sex hormones w E 2雌激素 w Testosterone睪固酮 w Hepatic encephalopathy 肝腦症 w Central fatigue中樞疲勞 w Intracerebral hemorrhage顱內出血 w Physical compression物理性壓迫 w Chemical stimulation化學性刺激 w 3 D reconstruction of tissue w Urethra (female rat)
常用的研究方法 w Morphological methods w Histological staining組織化學染色 w IHC免疫組織化學染色 w Neuronal tracing神經順向或逆向追蹤 w Intracellular dye injection細胞內染料注射 w Electrophysiological methods w Intracellular recording (current clamp)細胞內電生理 w Behavioral methods w Rota-rod test滾輪測試 w Weight-loaded forced swimming test負重游泳測試 w Activities test活動力測試 w Morris water maze水迷宮
Rota-rod test Weight-loaded forced swimming test Morris water maze
Topics w Stem cells幹細胞 w Intracellular electrophysiological recording 細胞內電生理 w Patch clamp w Current clamp w Field potential recording w Immunohistochemical (IHC) staining免疫 組織化學染色 w Culture w IHC-F, IHC-P w WB
Section I The application of stem cells 7
Regeneration K. Poss, Science, 2002 L. Iten, 1973 P. Anversa, NEJM, 2002
Stem cells w Features w Self-renewal自我更新 w Potency分化潛能 w Sources w Embryonic stem cells w Adult stem cells w Classification w Totipotent stem cells全能幹細胞 w Pluripotent stem cells多功能幹細胞 w Multipotent stem cells多潛能幹細胞 w Unipotent stem cells專一性幹細胞
The sources of Pluripotent stem cells w Inner cell mass http: //php. med. unsw. edu. au/embryology/index. php?
The sources of stem cells w w w w 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 (i. PS) 誘導多功能幹細胞
Embryonic stem cell Type Adult stem cell Embryonic stem cells are pluripotent Adult stem cells are limited to differentiating Culture Embryonic stem Adult stem cells grown easily are rare in mature in culture tissues Transplant rejection Embryonic don't yet Adult stem know cells, less likely to initiate rejection
The Applications of Stem cells w 細胞、組織、器官修補更新 w 腦中風、中樞神經退化 w 心衰竭… w 人造器官與組織的來源 w 新藥開發 w 基因功能研究 w 基因治療的 具 w 毒理、藥理研究 w 癌症研究
Stem Cell Therapy M. Mimeault, Stem Cell, 2006
Stem Cells in Cardiac Disorders The spotlight of regenerative medicine w Target disorder: myocardial infarction M. Schneider, Nature, 2004 A. Mathur, Lancet, 2004
The Applications of Stem cells w 細胞、組織、器官修補更新 w 人造器官與組織的來源 w 外耳 w 心臟 w 新藥開發 w 基因功能研究 w 基因治療的 具 w 毒理、藥理研究 w 癌症研究
人造心臟
Section II Electrophysiological properties of the neurons 19
Basic Concepts Ø Volt ØA charge difference between two points in space Ø Ions – charged particles Ø Anions – Negatively charged particles Ø ClØ Cations – Positively charged particles Ø Na+, Ca 2+, K+ 20
Basic Concepts Forces that determine ionic movement Ø Electrostatic forces靜電的力量 Ø同性相斥,異性相吸 Ø Concentration forces濃度 的力量 ØDiffusion 擴散 ØOsmosis 反滲透 21
Calculating equilibrium potential Nernst Equation Ø Allows theoretical membrane potential to be calculated for particular ion. ØMembrane potential that would exactly balance the diffusion gradient and prevent the net movement of a particular ion. ØValue depends on the ratio of [ion] on the 2 sides of the membrane. 22
Nernst equation能斯特方程式 Equilibrium potential 平衡電位(m. V) , Eion [C]o RT = ln z. F [C]i [C]o R = T = F = and [C]i = extra and intracellular [ion] Universal gas constant氣體常數(8. 3 joules. K-1. mol-1) Absolute temperature 絕對溫度(°K) 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, Ca 2+ = +2, Cl- = -1) For K+, with [K+]o = 4 mmol. l-1 and [K+]i = 150 mmol. l-1 At 37°C, EK = -97 m. V, ENa = +60 m. V, ECl = -67 m. V 23
Selective Permeability of Membranes ØSome ions permitted to cross more easily than others ØNeuronal membranes contain ion channels ØProtein tubes that span the membrane ØSome stay open all the time (nongated) ØSome open on the occasion of an action potential, causing a change in the permeability of the membrane (gated) 24
Resting Membrane Potential靜止膜電 位 ØNa+ and Cl- are more concentrated outside the cell ØK+ and organic anions (organic acids and proteins) are more concentrated inside. 25
The Sodium-Potassium Pump extrudes Na+ from the cell while taking in K + 26
The formation of resting potential Ø Concentration difference of K+ across the membrane Ø Permeability of Na+ and K+ during the resting state Ø Na+-K+ pump Resting membrane potential (RMP) 27
Intracellular vs extracellular ion concentrations Ion Intracellular Extracellular Na+ K+ Mg 2+ Ca 2+ H+ 5 -15 m. M 140 m. M 0. 5 m. M 10 -7. 2 M (p. H 7. 2) 145 m. M 1 -2 m. M 10 -7. 4 M (p. H 7. 4) Cl- 5 -15 m. M 110 m. M 28
Resting Membrane Potential Ø 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 m. V Ø Resting membrane potential of most cells ranges from - 65 to – 85 m. V. 29
Basic Electrophysiological Terms Ø Polarization極化: a state in which membrane is polarized at rest, negative inside and positive outside. Ø Depolarization去極化: the membrane potential becomes less negative than the resting potential (close to zero). Ø Hyperpolarization再極化: the membrane potential is more negative than the resting level. 30
Action Potential動作電位 Successive Stages: (2) (1) Resting Stage (3) (2) Depolarization stage (1 ) (4) (3) Repolarization stage (4) After-potential stage 31
Ion Permeability during the AP 32 Figure 8 -12: Refractory periods
Electrophysiological Method to Record Membrane Potential I Voltage Clamp電壓箝制 Current Clamp電流箝制 33
The Nobel Prize in Physiology or Medicine (1963) Alan Lloyd Hodgkin Andrew Fielding Huxley “for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane” 34
The Hodgkin-Huxley Model of Action Potential Generation 35
Modern proof of nature of currents Use ion selective agents -河魨毒(TTX) -四乙胺 (TEA) 36
Intracellular electrophysiological recording
Membrane properties Spike generating patterns Action potential Current– voltage Relationships V=I*R
Electrical signals between neurons
Reversal potential 反轉動位 Excitatory Post-Synaptic Potential Inhibitory Post-synaptic Potential
Electrophysiological Method to Record Membrane Potential II Patch Clamp膜片箝制 41
The Nobel Prize in Physiology or Medicine (1991) Erwin Neher Bert Sakmann "for their discoveries concerning the function of single ion channels in cells" 42
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
How channel conductances accumulate 45
Whole-cell recording
Electrophysiological Method to Record Membrane Potential III Extracellular recording 細胞外記錄 (Field potential) 47
Extracellular recording Field Excitatory Post-synaptic Potential (f. EPSP) EPSP LTP stimulate induced by a short tetanus (10 pulses at 100 Hz) at hippocampal CA 1 synapses Synaptic potential allow transmission of information from one neuron to another
Procedures of electrophysiological studies w Decapitated w Vibratome section w Culture w Recording
Vibratome section
Conservation of brain slice activity Artificial cerebrospinal fluid人 腦脊髓液 (ACSF) Na. Cl, KCl, Ca. Cl, Mg. Cl 2, Na. HCO 3 Na. H 2 PO 4, glucose Energy Glucose + 95% Oxygen and 5% CO 2 Slice preparation 4℃ in ACSF (without Ca 2+) Slice maintenance 25℃ in ACSF
Patch clamp assembly digitizer analog‐to‐digital converter amplifier 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: (1 a) microbeaker; (1 b) artificial extracellular fluid; (1 c) microstir bar; (1 d) lipid monolayer; (1 e) lipid bilayer; (1 f) patch pipette; (1 g) artificial intracellular fluid; (1 h) recording electrode; (1 i) reference electrode; (1 j) electrode holder; (1 k) 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/S 007668790617007 X
Current clamp
Copper mesh銅網
Micropipette Puller
Extracellular recordings with Microelectrode arrays微電極陣列
Section III Immunohistochemical (IHC) staining Antibody w Polyclonal antibody多株抗體 w Monoclonal antibody單株抗體 57
IHC Staining Protocol w Fixation w 4% paraformaldehyde w Transcardial perfusion w antigen retrieval protocols w Section w Paraffin section w Cryosection w Antigen - Antibody reaction (Temperature) w Enzymes and Chromogens
Transcardial Perfusion
Antigen Retrieval抗原還原 w During the process of formalin fixation, many antigenic sites are ‘masked’ and are therefore sometimes difficult or impossible to stain without antigen retrieval. w 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. w Commonly used proteolytic enzymes include trypsin, pepsin and protease. 56
Antigen Retrieval w Heat induced epitope retrieval (HIER) includes microwaving, pressure cooking, steaming, autoclaving or using the Pre. Treatment Module™. w Requires buffer of different concentrations and p. H. Commonly used buffers include w Citrate buffter at p. H 6. 0 w EDTA at p. H 8. 0 w Tris-HCL at p. H 10. 0 57
Antigen Retrieval Citrate buffer, p. H 6. 0 No Antigen Retrieval These photos show the staining results of CD 3 antibody on tonsil, with and without antigen retrieval. 58
Paraffin section
Cryosection
IHC Staining Methods w Direct Method w Indirect Method w Two-Step w Three-Step w Avidin-Biotin Complex (ABC) Method 69
Direct Method primary Ab Enzyme or fluorescent antigen It has the advantages of rapidity, ease of performance and limited nonspecific reaction. 73
Direct IHC Staining Adiponectin in Mouse skin E-Cadherin and DAPI
Indirect Method w Uses an enzyme-labeled secondary antibody that is directed against the unlabeled primary antibody. w If the primary antibody (which is now the antigen) is made in mouse, the secondary antibody must be against mouse immunoglobulin. w 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 - Procedure primary Ab (mouse) antigen An unlabeled primary antibody binds to the tissue antigen. 77
Two-Step Indirect Method secondary Ab (rabbit anti-mouse) Enzyme or fluorescent An enzyme-labeled secondary antibody binds to the primary antibody. 78
Indirect IHC Staining CD 133 antibody (Stem Cell Marker) and DAPI
Multiple Immunolabeling
Multiple immunofluorescence labeling of fixed sections of breast cancer A 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 MAP 2 (green) and GFAP (red). Nuclei of all cells are stained with Hoechst dye (blue). http: //www. abcam. com/
Small intestine stained with anti-CD 10 and DAB+Ni substrate (gray/black). Cytokeratin 20 is visualized with Imm. PRESS™ Anti-Mouse Ig and Vector® Nova. RED™ substrate (red) http: //www. abcam. com/
Three-Step Indirect Method An enzyme-labeled tertiary antibody is added and binds to the secondary antibody. tertiary Ab (goat anti-rabbit) 85
Avidin-Biotin Methods w Uses the strong and high affinity of avidin (egg white glycoprotein) for biotin (water-soluble vitamin). w Avidin has four binding sites for biotin but fewer than four molecules of biotin will actually bind to avidin 100 biotin molecules
Avidin-Biotin Methods w Two of the most common methods include w Avidin-Biotin enzyme Complex (ABC) w Labeled Strept. Avidin-Biotin (LSAB) w The inherent amplification of sensitivity offered by avidin and biotin makes these methods more favorable than PAP or APAAP. w Streptavidin, a bacterial protein has recently replaced avidin because it produces less background staining than avidin. 101
ABC Method w The enzyme complex is prepared by mixing biotinylated enzyme (HRP or AP) and avidin. biotinylated enzyme avidin-biotin-enzyme complex q This preformed avidin-biotin-enzyme complex then reacts with the biotinylated secondary antibody. 103
ABC - Procedure An unlabeled primary antibody binds to the antigen 104 primary Ab (mouse)
ABC - Procedure A biotinylated secondary antibody binds to the primary antibody. secondary Ab (rabbit anti-mouse) biotin 105
ABC - Procedure A preformed avidin-biotin-enzyme complex solution is added and binds to the biotinylated secondary antibody. avidin-biotin-enzyme complex 106
ABC - Procedure A substrate-chromogen solution is added ending the reaction and producing a colored end-product. substratechromogen 107
LSAB Method w Uses enzyme-conjugated streptavidin. Streptavidin is conjugated to several molecules of enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP). w The secondary antibody is conjugated to numerous biotin molecules, each of which can potentially bind to an enzyme-conjugated streptavidin. 109
LSAB – Procedure An unlabeled primary antibody binds to tissue antigen. primary Ab antigen 110
LSAB – 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 secondary Ab biotin
LSAB – Procedure An enzyme-labeled streptavidin is added and binds to the secondary antibody. HRP-streptavidin 112
LSAB – Procedure A substrate-chromogen solution is added producing a colored end-product. substrate- chromogen 113
Enzymes and Chromogens w Detection systems attach enzyme labels to primary or secondary antibodies to visualize the localized antibody-antigen binding in tissue section. w 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. w A chromogen is a substance that absorbs light, producing color. 60
Enzymes and Chromogens w Commonly used enzyme labels for IHC procedures include w horseradish peroxidase (HRP)過氧化物酶 w alkaline phosphatase (AP)鹼性磷酸酶 w HRP is an enzyme that catalyze the reduction of hydrogen peroxide (H 2 O 2) to water and oxygen. w Commonly used chromogens for HRP include w 3 -amino-9 -ethylcarbazole (AEC) w 3, 3’-diaminobenzidine (DAB) 61
AEC w AEC is oxidized by HRP producing a bright red reaction product. This reaction product is not stable and may fade over time. C 2 H 5 N NH 2 Structure of AEC 62
DAB w DAB is oxidized by HRP producing a dark brown reaction product. This reaction product is stable and does not fade over time. +H +H 3 N 4 Cl - NH 3+ 3 N NH 3+ Structure of DAB 64
AEC and DAB AEC chromogen Mart-1 Melanoma DAB chromogen Mart-1 Melanoma Examples of staining results using AEC and DAB chromogens. 66
Stained Pattern - Cell Membrane c-erb. B-2 Breast carcinoma AEC chromogen 136 CD 3/T-Cell Tonsil AEC chromogen
Stained Pattern - Nuclear Estrogen Receptor (ER) Breast carcinoma AEC chromogen 137 Cyclin D 1 Mantle cell lymphoma DAB chromogen
Stained Pattern - Cytoplasmic Keratin, Pan Colon carcinoma AEC chromogen 138 Vimentin Melanoma AEC chromogen
Stem cells w Features w Self-renewal自我更新 w Potency分化潛能 w Sources w Embryonic stem cells w Adult stem cells w Classification w Totipotent stem cells全能幹細胞 w Pluripotent stem cells多功能幹細胞 w Multipotent stem cells多潛能幹細胞 w Unipotent stem cells專一性幹細胞
The Nobel Prize in physiology or medicine 2012 w Gurdon JB w Shinya Yamanaka(山中伸彌) w Induced pluripotent stem cells (i. PS) 誘 導多功能幹細胞
Resting Membrane Potential Ø 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 m. V Ø Resting membrane potential of most cells ranges from - 65 to – 85 m. V. 100
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 腦脊髓液 (ACSF) Na. Cl, KCl, Ca. Cl, Mg. Cl 2, Na. HCO 3 Na. H 2 PO 4, glucose Energy Glucose + 95% Oxygen and 5% CO 2 Slice preparation 4℃ in ACSF (without Ca 2+) Slice maintenance 25℃ in ACSF
IHC Staining Protocol w Fixation w 4% paraformaldehyde w Transcardial perfusion w antigen retrieval protocols w Section w Paraffin section w Cryosection w Antigen - Antibody reaction (Temperature) w Enzymes and Chromogens
Direct Method primary Ab Enzyme or fluorescent antigen It has the advantages of rapidity, ease of performance and limited nonspecific reaction. 73
Avidin-Biotin Methods w Two of the most common methods include w Avidin-Biotin enzyme Complex (ABC) w Labeled Strept. Avidin-Biotin (LSAB) w The inherent amplification of sensitivity offered by avidin and biotin makes these methods more favorable than PAP or APAAP. w Streptavidin, a bacterial protein has recently replaced avidin because it produces less background staining than avidin. 101
Enzymes and Chromogens w Commonly used enzyme labels for IHC procedures include w horseradish peroxidase (HRP)過氧化物酶 w alkaline phosphatase (AP)鹼性磷酸酶 w HRP is an enzyme that catalyze the reduction of hydrogen peroxide (H 2 O 2) to water and oxygen. w Commonly used chromogens for HRP include w 3 -amino-9 -ethylcarbazole (AEC) w 3, 3’-diaminobenzidine (DAB) 61
Polyclonal Antibody Production w A rabbit is injected subcutaneously with a purified dose of antigen. w The rabbit’s immune system responds by producing antibodies specific to the injected antigen. w Blood is harvested from the ear at the peak of antibody production. w Red blood cells and clotting proteins are removed and the antiserum is purified. Y Y Y 25 Y Y Y
Polyclonal Antibodies Polyclonal antibodies reacting with various epitopes Each antibody is made by a different B-cell 22
Polyclonal Antibody Production w Polyclonal antibodies are purified either by Protein Purification or Antigen Affinity Chromatography. w Protein Purification eliminates the bulk of serum proteins but does not eliminate nonspecific immunoglobulin fraction. w 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 Antibodies w Monoclonal antibodies are derived from a single B-cell and are produced as a single class of immunoglobulin. w They are raised by fusion of the specific B-cells with immortal myeloma (B-cell) cancer cells to form a hybridoma. w A hybridoma can multiply indefinitely and continuously produce a specific monoclonal antibody. w They react with a specific epitope on a given antigen, giving less background staining. 30
Monoclonal Antibody Production w A mouse is injected subcutaneously with a purified dose of antigen. w The mouse’s immune system responds by producing antibodies specific to the injected antigen. 34
Monoclonal Antibody Production spleen B-lymphocytes The antibody-producing B-cells are harvested from the spleen or lymph nodes. 36
Monoclonal Antibody Production myeloma cells B-lymphocytes The B-cells are fused with mouse myeloma cells forming immortal hybrid cells or hybridomas. 37
Monoclonal Antibody Production The generated hybridomas will produce many copies of the exact same antibody. 38
Monoclonal Antibody Production w. The hybridomas: w. Transplanted into the peritoneal cavity w. Propagated in a tissue culture medium 39
Monoclonal Antibodies Monoclonal antibodies reacting with similar epitopes 32
Antibody Titer and Dilution 1: 50 1: 400 47 1: 100 1: 800 1: 200
Incubation Time w Incubation time is inversely proportional to antibody concentration. Higher concentration of antibody allows shorter incubation time. w It can be from minutes to hours, with 30 -60 minutes the most common practice. 52
Incubation Temperature w Antibody-antigen reaction is hastened at 37 C as compared to room temperature. An increase in temperature also allows for a higher dilution of the antibody. 54 w Humidity chambers must be used when incubating at higher temperature to prevent drying of tissue sections.
f18274bf0cc737592808a19f268fc305.ppt