獸醫基礎科學概論 － 形態學 － 陳建榮 the role of morphological studies in research

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

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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

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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)

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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

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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.

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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.

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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)

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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

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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.

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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

100

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.

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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.

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