HISTOTECHNIQUE HISTOTECHNIQUE IN IN THE RESEARCH OF NEUROSCIENCE THE RESEARCH OF NEUROSCIENCE All images in this document is All images in this document is removed due to copyright removed due to copyright restriction restriction Ahmad Aulia Jusuf Ahmad Aulia Jusuf MD, PhD MD, PhD Department of Histology Department of Histology Faculty of Medicine University of Indonesia Faculty of Medicine University of Indonesia 2006 2006
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HISTOTECHNIQUE IN THE RESEARCH OF NEUROSCIENCE All images in this document is removed due to copyright restriction Ahmad Aulia Jusuf MD, PhD Department.
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HISTOTECHNIQUEHISTOTECHNIQUEIN IN
THE RESEARCH OF NEUROSCIENCETHE RESEARCH OF NEUROSCIENCEAll images in this document is All images in this document is
removed due to copyright removed due to copyright restrictionrestriction
Ahmad Aulia JusufAhmad Aulia Jusuf MD, PhD MD, PhDDepartment of HistologyDepartment of Histology
Faculty of Medicine University of IndonesiaFaculty of Medicine University of Indonesia20062006
Histoteknik/AAJ/Histologi-FKUI/2006Histoteknik/AAJ/Histologi-FKUI/2006 22Tuesday, June 27, 2006Tuesday, June 27, 2006
AgendaAgenda IntroductionIntroduction The techniqueThe techniquess in histology in histology
used in the research of used in the research of neuroscience neuroscience Tissue preparationTissue preparation StainingStaining
Routine stainingRoutine staining Special stainingSpecial staining
o Histochemical stainingHistochemical stainingo Immunocytochemical stainingImmunocytochemical stainingo In situ hybridizationIn situ hybridizationo Tunnel AssayTunnel Assay
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Introduction Histology no longer deals Histology no longer deals
merely with the structure of merely with the structure of bodybody But it also deals with its function But it also deals with its function
In fact the subject matter of In fact the subject matter of histology has a direct histology has a direct relationship to other relationship to other disciplinesdisciplines It is essential for their It is essential for their
understandingunderstanding
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Introduction
RememberRemember No function without structureNo function without structure All of the research in the All of the research in the
neuroscience has tight neuroscience has tight correlation with the structure correlation with the structure of tissue or organ as their of tissue or organ as their basicbasic
Researches in the Researches in the neuroscience need the neuroscience need the participation of Histologyparticipation of Histology
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Introduction
Histotechniques are the Histotechniques are the most important methods in most important methods in histology to visualize histology to visualize not only the structure of tissue not only the structure of tissue
or organor organ but also can be used in to but also can be used in to
study the location of the study the location of the expression of the gene, protein expression of the gene, protein and other molecules in the cell and other molecules in the cell or tissueor tissue
Histoteknik/AAJ/Histologi-FKUI/2006Histoteknik/AAJ/Histologi-FKUI/2006 66Tuesday, June 27, 2006Tuesday, June 27, 2006
Methods in histologyMethods in histology To visualize the structure of tissueTo visualize the structure of tissue Aims to study Aims to study
o The micro-architecture of tissueThe micro-architecture of tissueo The development of tissue or organ The development of tissue or organ
(histo-dynamic)(histo-dynamic)o The present of special molecules in the The present of special molecules in the
cell or tissuescell or tissueso The expression of gene in the tissueThe expression of gene in the tissueo The present and localization of protein The present and localization of protein
in the tissuein the tissueo The apoptosis process in the tissueThe apoptosis process in the tissue
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MixMixo Injection and inhalationInjection and inhalation
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Tissue Processing Tissue Processing
Tissue isolation and FixationTissue isolation and Fixation FixationFixation
Treatment of tissues with chemical agentTreatment of tissues with chemical agent The aims of fixationThe aims of fixation
o To preserve the microstructure of tissue as To preserve the microstructure of tissue as the living state and prevent the alteration of the living state and prevent the alteration of tissuetissue
o To prevent the autolysisTo prevent the autolysiso To prevent of bacterial digestionTo prevent of bacterial digestiono To harden the tissue and making it to be cut To harden the tissue and making it to be cut
easilyeasilyo To change the consistency of tissue from sol To change the consistency of tissue from sol
(soft) to become gel (hard)(soft) to become gel (hard)o To increase the optical differentiationTo increase the optical differentiationo Preparing for stainingPreparing for staining
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Tissue Processing Tissue Processing
DehydrationDehydration The process to The process to
remove the waterremove the water By using a graded By using a graded
series of alcohol or series of alcohol or other dehydration other dehydration agent (sucrose, agent (sucrose, ect)ect)
the tissue can be the tissue can be filled with the filled with the paraffin or other paraffin or other embedding agent embedding agent
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Tissue Processing Tissue Processing
ClearingClearing The process of The process of
tissue to prepare it tissue to prepare it for embeddingfor embedding
By using the By using the chemical that is chemical that is miscible with the miscible with the embedding agentembedding agent ChloroformChloroform Benzene / benzolBenzene / benzol Xylene / xylolXylene / xylol Cedar wood oilCedar wood oil Benzyl benzoatBenzyl benzoat Methyl benzoatMethyl benzoat
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Tissue Processing Tissue Processing
EmbeddingEmbedding The process to The process to
distinguish the distinguish the overlapping cells in a overlapping cells in a tissue and extra-tissue and extra-cellular matrix from cellular matrix from one anotherone another By embedding the By embedding the
tissue in the proper tissue in the proper medium such as medium such as paraffin or otherparaffin or other
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Tissue Processing Tissue Processing
EmbeddingEmbedding The methodThe method
The tissue is placed The tissue is placed in a suitable in a suitable container of melted container of melted paraffin or other paraffin or other until it is completely until it is completely infiltrated and infiltrated and become harderbecome harder
o forming a block forming a block containing the containing the tissue tissue
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The process to cut the specimen block in The process to cut the specimen block in the desired thicknessthe desired thickness
The methodThe method By using microtomeBy using microtome
o The machine is equipped with the blade and an The machine is equipped with the blade and an arm that advance the tissue block in specific arm that advance the tissue block in specific equal incrementsequal increments
o For light microscope : 5-10 micrometerFor light microscope : 5-10 micrometer By using cryostatBy using cryostat
o The block tissue is sectioned at subzero The block tissue is sectioned at subzero temperature using a pre-cooled steel bladetemperature using a pre-cooled steel blade
The sections are placed on pre-cooled glass The sections are placed on pre-cooled glass slides slides
Stained with specific dyesStained with specific dyes
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Tissue Processing Tissue Processing
MountingMounting The process to The process to
placed/mount the placed/mount the tissue sections on tissue sections on the adhesive the adhesive coated glass coated glass slidesslides Albumin Albumin White part of an White part of an
eggegg GelatinGelatin TespaTespa
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Staining Staining DefinitionDefinition
The process to stain the tissue sections The process to stain the tissue sections with the staining dyeswith the staining dyes
2 group2 group Routine stainingRoutine staining
Hematoxylin EosinHematoxylin Eosin Specific stainingSpecific staining
The most commonly used staining in The most commonly used staining in histologyhistology Cheapest stainingCheapest staining Easy to be doneEasy to be done The microstructure is stained wellThe microstructure is stained well Can be studied under light microscopyCan be studied under light microscopy
Hematoxylin-eosin stainingHematoxylin-eosin staining The nuclei of cells are stained blueThe nuclei of cells are stained blue (basophilic) (basophilic) in in
colorcolor The cytoplasms of cells The cytoplasms of cells including the organelles including the organelles
and other molecule and other molecule are stained pinkare stained pink (Acidophilic) (Acidophilic) in colorin color
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The staining is used to localize The staining is used to localize specific chemical constituents of specific chemical constituents of tissues and cellstissues and cells
Based on Based on enzyme activityenzyme activity
o Example : Nitric Oxide Synthesize (NOS) Example : Nitric Oxide Synthesize (NOS)
Chemical reactivityChemical reactivityo Example : Periodic acid Schiff (PAS) to Example : Periodic acid Schiff (PAS) to
localized the glycogen and carbohydrate rich localized the glycogen and carbohydrate rich moleculesmolecules
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A technique to identify cellular or A technique to identify cellular or tissue constituents (antigen) tissue constituents (antigen)
o By means of antigen-antibody interactionsBy means of antigen-antibody interactionsThe site of antibody bindingThe site of antibody binding
o Is identified by direct labeling of antibody or Is identified by direct labeling of antibody or by used a secondary labeling methodby used a secondary labeling method
The termsThe termso If tissue ImmunohistochemistryIf tissue Immunohistochemistryo If cell ImmunocytochemistryIf cell Immunocytochemistry
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A protein or carbohydrate or lipid A protein or carbohydrate or lipid moleculemolecule
o Has one or more antibody binding sitesHas one or more antibody binding sitesA highly specific topographical regions A highly specific topographical regions
composed of a small number of amino composed of a small number of amino acids or monosaccharide unit known as acids or monosaccharide unit known as antigenic determinant groups or antigenic determinant groups or epitopesepitopes
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Staining Staining In Situ HybridizationIn Situ Hybridization
A histological technique used to A histological technique used to determine determine the regions in the tissue or the cell the regions in the tissue or the cell
types within the organtypes within the organo Express a specific gene at light microscopic Express a specific gene at light microscopic
levellevel
2 types2 typesWhole mount insitu hybridizationWhole mount insitu hybridizationSection insitu hybridizationSection insitu hybridization
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Staining Staining In Situ HybridizationIn Situ Hybridization
The principlesThe principles The target m RNA can be hybridized with The target m RNA can be hybridized with
ribonucleic probe ribonucleic probe o Has been labeled previously with auto radio Has been labeled previously with auto radio
isotope or chemical agent isotope or chemical agent A peroxidase/alkaline phosphatase conjugated A peroxidase/alkaline phosphatase conjugated
antibody antibody o used to bind to the ribonucleic probeused to bind to the ribonucleic probe
A chemiluminescence detection systemA chemiluminescence detection systemo Used to detect the location of hybridization Used to detect the location of hybridization
By using the a substrate that give the specific By using the a substrate that give the specific color after reacting with antibodycolor after reacting with antibody
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Staining Staining In Situ HybridizationIn Situ Hybridization
RNA
Chemical agent labeled riboprobe
Alkaline phophatase conjugated antibody
Substrate
Specific color
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Staining Staining
In Situ HybridizationIn Situ Hybridization
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Staining Staining
Tunel AssayTunel AssayA technique used to identified A technique used to identified
programmed cell death (apoptosis) programmed cell death (apoptosis) By specific labeling of nuclear DNA By specific labeling of nuclear DNA
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Staining Staining
Programmed Cell DeathProgrammed Cell DeathA selective of physiological cell A selective of physiological cell
deletion deletion plays a major role in developmental plays a major role in developmental
biology and in maintenance of the steady biology and in maintenance of the steady state in continuously renewing tissue state in continuously renewing tissue
o By controlling the balance cell replication and By controlling the balance cell replication and cell death cell death
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Staining Staining
Tunnel AssayTunnel Assay The principlesThe principles
Based on the specific binding of terminal Based on the specific binding of terminal deoxynucleotide transferase (TdT) to 3’-OH deoxynucleotide transferase (TdT) to 3’-OH ends of broken DNA fragment ensuming a ends of broken DNA fragment ensuming a synthesis of a polydeoxynucleotide polymersynthesis of a polydeoxynucleotide polymer
TdT was used to incorporate biotinylated TdT was used to incorporate biotinylated deoxyuridine at sites of DNA breaksdeoxyuridine at sites of DNA breaks
The signal then amplified by avidin peroxidase, The signal then amplified by avidin peroxidase, enabling conventional histochemical enabling conventional histochemical identification by light microscopyidentification by light microscopy
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