HISTOTECHNIQUE IN THE RESEARCH OF NEUROSCIENCE All images in this document is removed due to copyright restriction Ahmad Aulia Jusuf MD, PhD Department of Histology Faculty of Medicine University of Indonesia 2006

Ahmad Aulia Jusuf/Histologi-FKUI Tuesday, June 27, 2006 Agenda Introduction The techniques in histology used in the research of neuroscience Tissue preparation Staining Routine staining Special staining Histochemical staining Immunocytochemical staining In situ hybridization Tunnel Assay Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006 Histoteknik/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Introduction Histology no longer deals merely with the structure of body But it also deals with its function In fact the subject matter of histology has a direct relationship to other disciplines It is essential for their understanding Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Introduction Remember No function without structure All of the research in the neuroscience has tight correlation with the structure of tissue or organ as their basic Researches in the neuroscience need the participation of Histology Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Introduction Histotechniques are the most important methods in histology to visualize not only the structure of tissue or organ but also can be used in to study the location of the expression of the gene, protein and other molecules in the cell or tissue Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Introduction Definition Histotechniques Methods in histology To visualize the structure of tissue Aims to study The micro-architecture of tissue The development of tissue or organ (histo-dynamic) The present of special molecules in the cell or tissues The expression of gene in the tissue The present and localization of protein in the tissue The apoptosis process in the tissue Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Introduction Histotechnique Tissue processing Staining Routine Special Histochemical staining Immunocytochemical staining In situ hybridization Tunnel Assay Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing The sequencing methods to conserve and prepare the tissue for studying Their natural Their living state The steps of tissue processing Tissue Isolation Fixation Dehydration Clearing Impregnation Embedding Blocking Cutting/Section Mounting Staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing The steps of tissue processing Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Resource for tissue Cadaver Ideal Difficult to find Animal monkey goat pig cat dog mice rabbit Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Preparation Tools minor surgery, perfusion machine, bottles, plates, histological slide, jar, tissue bath, microtome, labeling tools Solution Fixative Dehydration agent Clearing agent Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Preparation Samples Cadaver Animal Monkey The arrangement and composition of tissue is very similar to human Goat Purkinje fibers Pig Classic hepatic lobule, Hulsen artery in the lien Cat and dog Rabbit Foliate papil Other Paraffin Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Tissue isolation and Fixation Anesthetic Injection Phenobarbital, chloraldehyde Inhalation Ether Mix Injection and inhalation Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Tissue isolation and Fixation Fixation Treatment of tissues with chemical agent The aims of fixation To preserve the microstructure of tissue as the living state and prevent the alteration of tissue To prevent the autolysis To prevent of bacterial digestion To harden the tissue and making it to be cut easily To change the consistency of tissue from sol (soft) to become gel (hard) To increase the optical differentiation Preparing for staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Tissue isolation and Fixation Fixation The most important step in tissue processing failed in fixation means failed in the whole tissue processing can not be repeated Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Tissue isolation and Fixation Method Supravital/intra vital fixation Fixation is done in the living state Immersed Fixation Tissues are immersed in the fixative agent after isolating Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Tissue isolation and Fixation Fixation The fixative agent depends to the purpose and staining solution fixative solution formaline 10% , Muller, Bouin, picric acid, Zenker, mercury chloride, ethyl alcohol, acetic acid Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Dehydration The process to remove the water By using a graded series of alcohol or other dehydration agent (sucrose, ect) the tissue can be filled with the paraffin or other embedding agent Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Clearing The process of tissue to prepare it for embedding By using the chemical that is miscible with the embedding agent Chloroform Benzene / benzol Xylene / xylol Cedar wood oil Benzyl benzoat Methyl benzoat Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Embedding The process to distinguish the overlapping cells in a tissue and extra-cellular matrix from one another By embedding the tissue in the proper medium such as paraffin or other Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Embedding The method The tissue is placed in a suitable container of melted paraffin or other until it is completely infiltrated and become harder forming a block containing the tissue Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Sectioning The process to cut the specimen block in the desired thickness The method By using microtome The machine is equipped with the blade and an arm that advance the tissue block in specific equal increments For light microscope : 5-10 micrometer By using cryostat The block tissue is sectioned at subzero temperature using a pre-cooled steel blade The sections are placed on pre-cooled glass slides Stained with specific dyes Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Sectioning Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Tissue Processing Mounting The process to placed/mount the tissue sections on the adhesive coated glass slides Albumin White part of an egg Gelatin Tespa Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Definition The process to stain the tissue sections with the staining dyes 2 group Routine staining Hematoxylin Eosin Specific staining Histochemistry Immunocytochemistry Insitu hybridization Tunnel Assay Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Routine staining The most commonly used staining in histology Cheapest staining Easy to be done The microstructure is stained well Can be studied under light microscopy Hematoxylin-eosin staining The nuclei of cells are stained blue (basophilic) in color The cytoplasms of cells including the organelles and other molecule are stained pink (Acidophilic) in color Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Hematoxylin-eosin staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Histochemistry staining The staining is used to localize specific chemical constituents of tissues and cells Based on enzyme activity Example : Nitric Oxide Synthesize (NOS) Chemical reactivity Example : Periodic acid Schiff (PAS) to localized the glycogen and carbohydrate rich molecules Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Histochemistry staining Reactions of interest monitored by formation of an insoluble precipitate that take a certain color Staining can be applied to the whole mount or histological slide Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Histochemistry staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Definition A technique to identify cellular or tissue constituents (antigen) By means of antigen-antibody interactions The site of antibody binding Is identified by direct labeling of antibody or by used a secondary labeling method The terms If tissue Immunohistochemistry If cell Immunocytochemistry Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Antigens A protein or carbohydrate or lipid molecule Has one or more antibody binding sites A highly specific topographical regions composed of a small number of amino acids or monosaccharide unit known as antigenic determinant groups or epitopes Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Antibody A molecule that can recognize and bind to the antigen 2 type of antibody Polyclonal antibody An antibody that can bind to several similar epitopes May cross react with other molecule Low sensitivity Monoclonal antibody An antibody which is sensitive only to one epitope Not cross react with other molecule High sensitivity Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Immunocyto/histochemical staining Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining In Situ Hybridization A histological technique used to determine the regions in the tissue or the cell types within the organ Express a specific gene at light microscopic level 2 types Whole mount insitu hybridization Section insitu hybridization Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining In Situ Hybridization The principles The target m RNA can be hybridized with ribonucleic probe Has been labeled previously with auto radio isotope or chemical agent A peroxidase/alkaline phosphatase conjugated antibody used to bind to the ribonucleic probe A chemiluminescence detection system Used to detect the location of hybridization By using the a substrate that give the specific color after reacting with antibody Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining In Situ Hybridization Alkaline phophatase conjugated antibody Chemical agent labeled riboprobe RNA Specific color Substrate Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining In Situ Hybridization Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Tunel Assay A technique used to identified programmed cell death (apoptosis) By specific labeling of nuclear DNA fragmentation TdT mediated dUTP biotin Nick End Labeling Method Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Programmed Cell Death A selective of physiological cell deletion plays a major role in developmental biology and in maintenance of the steady state in continuously renewing tissue By controlling the balance cell replication and cell death Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Tunnel Assay The principles Based on the specific binding of terminal deoxynucleotide transferase (TdT) to 3’-OH ends of broken DNA fragment ensuming a synthesis of a polydeoxynucleotide polymer TdT was used to incorporate biotinylated deoxyuridine at sites of DNA breaks The signal then amplified by avidin peroxidase, enabling conventional histochemical identification by light microscopy Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Tunnel Assay TdT deoxynucleotidyl transferase Avidin peroxidase conjugated biotinylated deoxyuridine Broken DNA polydeoxynucleotide polymer Specific color Substrate Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 Staining Tunnel Assay Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006

Histoteknik/AAJ/Histologi-FKUI/2006 thank you Tuesday, June 27, 2006 Histoteknik/AAJ/Histologi-FKUI/2006