HISTOTECHNIQUE IN THE RESEARCH OF NEUROSCIENCE All images in this document is removed due to copyright restriction Ahmad Aulia Jusuf MD, PhD Department

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


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


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


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


Introduction

DefinitionDefinition HistotechniquesHistotechniques

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


Introduction

HistotechniqueHistotechnique Tissue processingTissue processing StainingStaining

RoutineRoutine SpecialSpecial

o Histochemical stainingHistochemical stainingo Immunocytochemical stainingImmunocytochemical stainingo In situ hybridizationIn situ hybridizationo Tunnel AssayTunnel Assay


Tissue ProcessingTissue Processing The sequencing methodsThe sequencing methods

to conserve and prepare the to conserve and prepare the tissue for studying tissue for studying

Their natural Their natural Their living state Their living state

The steps of tissue processingThe steps of tissue processing Tissue IsolationTissue Isolation FixationFixation DehydrationDehydration ClearingClearing ImpregnationImpregnation Embedding Embedding BlockingBlocking Cutting/SectionCutting/Section MountingMounting StainingStaining


Tissue ProcessingTissue Processing The steps of tissue processingThe steps of tissue processing


Tissue ProcessingTissue Processing Resource for tissueResource for tissue

CadaverCadaver IdealIdeal Difficult to findDifficult to find

AnimalAnimal monkeymonkey goatgoat pigpig catcat dogdog micemice rabbitrabbit


Tissue Processing Tissue Processing

PreparationPreparationTools Tools

minor surgery, perfusion machine, minor surgery, perfusion machine, bottles, plates, histological slide, jar, bottles, plates, histological slide, jar, tissue bath, microtome, labeling toolstissue bath, microtome, labeling tools

SolutionSolutionFixativeFixativeDehydration agentDehydration agentClearing agentClearing agent


Tissue Processing Tissue Processing PreparationPreparation

SamplesSamples CadaverCadaver AnimalAnimal

o Monkey Monkey The arrangement and The arrangement and

composition of tissue is composition of tissue is very similar to humanvery similar to human

o GoatGoat Purkinje fibersPurkinje fibers

o PigPig Classic hepatic lobule, Classic hepatic lobule,

Hulsen artery in the lien Hulsen artery in the lien o Cat and dogCat and dogo RabbitRabbit

Foliate papilFoliate papil OtherOther

ParaffinParaffin



Tissue isolation and Fixation Tissue isolation and Fixation AnestheticAnesthetic

InjectionInjectiono Phenobarbital, chloraldehydePhenobarbital, chloraldehyde

InhalationInhalationo EtherEther

MixMixo Injection and inhalationInjection and inhalation



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


Tissue Processing Tissue Processing Tissue isolation Tissue isolation

and Fixationand Fixation FixationFixation

The most The most important step in important step in tissue processingtissue processing

o failed in fixation failed in fixation means failed in means failed in the whole tissue the whole tissue processingprocessing

o can not be can not be repeatedrepeated



Tissue isolation and Tissue isolation and FixationFixation

MethodMethodo Supravital/intra vitalSupravital/intra vital

fixationfixation Fixation is done in Fixation is done in

the living statethe living stateo Immersed FixationImmersed Fixation

Tissues are Tissues are immersed in the immersed in the fixative agent after fixative agent after isolatingisolating



Tissue isolation and Tissue isolation and FixationFixation FixationFixation

The fixative agentThe fixative agento depends to the purpose depends to the purpose

and staining solutionand staining solutiono fixative solutionfixative solution

formaline 10% , formaline 10% , Muller, Bouin, picric Muller, Bouin, picric acid, Zenker, mercury acid, Zenker, mercury chloride, ethyl chloride, ethyl alcohol, acetic acidalcohol, acetic acid



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



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



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



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


Tissue Processing Tissue Processing SectioningSectioning

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


Tissue Processing Tissue Processing SectioningSectioning



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


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

HistochemistryHistochemistry ImmunocytochemistryImmunocytochemistry Insitu hybridizationInsitu hybridization Tunnel AssayTunnel Assay


Staining Staining Routine stainingRoutine 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


Staining Staining Hematoxylin-eosin stainingHematoxylin-eosin staining


Staining Staining Histochemistry stainingHistochemistry staining

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



Reactions of interest Reactions of interest monitored by formation of an insoluble monitored by formation of an insoluble

precipitate that take a certain color precipitate that take a certain color Staining can be applied to the Staining can be applied to the

whole mount or histological slidewhole mount or histological slide




Staining Staining Immunocyto/histochemical Immunocyto/histochemical

stainingstainingDefinitionDefinition

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


Staining Staining Immunocyto/histochemical stainingImmunocyto/histochemical staining


Staining Staining Immunocyto/histochemical Immunocyto/histochemical

stainingstainingAntigensAntigens

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



AntibodyAntibody A molecule that can recognize and bind to the A molecule that can recognize and bind to the

antigenantigen 2 type of antibody2 type of antibody

o Polyclonal antibodyPolyclonal antibody An antibody that can bind to several similar An antibody that can bind to several similar

epitopesepitopes May cross react with other molecule May cross react with other molecule Low sensitivityLow sensitivity

o Monoclonal antibodyMonoclonal antibody An antibody which is sensitive only to one An antibody which is sensitive only to one

epitopeepitope Not cross react with other moleculeNot cross react with other molecule High sensitivityHigh sensitivity






Staining Staining

Immunocyto/Immunocyto/histochemical staininghistochemical staining


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



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



RNA

Chemical agent labeled riboprobe

Alkaline phophatase conjugated antibody

Substrate

Specific color


Staining Staining

In Situ HybridizationIn Situ Hybridization


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

fragmentationfragmentationTTdT mediated ddT mediated dUUTP biotin TP biotin NNick ick EEnd nd

LLabeling Methodabeling Method


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


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


Staining Staining Tunnel AssayTunnel Assay

Broken DNA

TdT deoxynucleotidyl transferase

Avidin peroxidase conjugated biotinylated biotinylated deoxyuridinedeoxyuridine

Substrate

Specific color

polydeoxynucleotidpolydeoxynucleotide polymere polymer


Staining Staining

Tunnel AssayTunnel Assay


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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