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獸醫基礎科學概論 - 形態學 - 陳建榮 獸醫基礎科學概論 - 形態學 - 陳建榮

The role of morphological studies in research 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 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 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 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 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 Section I The application of stem cells 7

Regeneration K. Poss, Science, 2002 L. Iten, 1973 P. Anversa, NEJM, 2002 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 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. 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 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 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 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 Cell Therapy M. Mimeault, Stem Cell, 2006

Stem Cells in Cardiac Disorders The spotlight of regenerative medicine w Target disorder: myocardial 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 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 人造心臟

Section II Electrophysiological properties of the neurons 19 Section II Electrophysiological properties of the neurons 19

Basic Concepts Ø Volt ØA charge difference between two points in space Ø Ions 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 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 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 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 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+ 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 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 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+ 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 + 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, 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 ) 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 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 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 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 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 Modern proof of nature of currents Use ion selective agents -河魨毒(TTX) -四乙胺 (TEA) 36

Intracellular electrophysiological recording Intracellular electrophysiological recording

Membrane properties Spike generating patterns Action potential Current– voltage Relationships V=I*R Membrane properties Spike generating patterns Action potential Current– voltage Relationships V=I*R

Electrical signals between neurons Electrical signals between neurons

Reversal potential 反轉動位 Excitatory Post-Synaptic Potential Inhibitory Post-synaptic Potential 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 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 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 Patch clamp recording

Inside out: The effects of intracellular molecular on receptor ion channel outside out: The 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 How channel conductances accumulate 45

Whole-cell recording Whole-cell recording

Electrophysiological Method to Record Membrane Potential III Extracellular recording 細胞外記錄 (Field potential) 47 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 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 Procedures of electrophysiological studies w Decapitated w Vibratome section w Culture w Recording

Vibratome section Vibratome section

Conservation of brain slice activity Artificial cerebrospinal fluid人 腦脊髓液 (ACSF) Na. Cl, KCl, Ca. 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, 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 Current clamp

Copper mesh銅網 Copper mesh銅網

Micropipette Puller Micropipette Puller

Extracellular recordings with Microelectrode arrays微電極陣列 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 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 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 Transcardial Perfusion

Antigen Retrieval抗原還原 w During the process of formalin fixation, many antigenic sites are ‘masked’ 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 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 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 Paraffin section

Cryosection Cryosection

IHC Staining Methods w Direct Method w Indirect Method w Two-Step w Three-Step w 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, 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 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 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 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 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 Indirect IHC Staining CD 133 antibody (Stem Cell Marker) and DAPI

Multiple Immunolabeling Multiple Immunolabeling

Multiple immunofluorescence labeling of fixed sections of breast cancer A formalin-fixed paraffin-embedded section of 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) 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 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 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) 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 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 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 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 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 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 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 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 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 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. 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 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 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 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 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 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 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 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 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 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 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) 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 + 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 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. 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 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, 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 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 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 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 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 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 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 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 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 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 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 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 Monoclonal Antibodies Monoclonal antibodies reacting with similar epitopes 32

Antibody Titer and Dilution 1: 50 1: 400 47 1: 100 1: 800 1: 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 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 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.