Histology Techniques PDF

Histology 1  Aims Provide an overview of a number of different histology techniques, highlighting relative strengths and weaknesses along with applications for these techniques. 1. Review basic components and types of tissue 2. Understand how to process tissue samples for h...

Histology 1  Aims Provide an overview of a number of different histology techniques, highlighting relative strengths and weaknesses along with applications for these techniques. 1. Review basic components and types of tissue 2. Understand how to process tissue samples for histological evaluation 3. Understand the different processing methods available 4. Overview of the principles behind staining techniques 5. Understanding the principles behind antibody labeling 6. Overview of microscopy techniques 2  Histology? The study of cellular structure and function of the body The anatomical study of the microscopic structure of tissues 1015 cells in the human body 200+ cell types in the body Four main types of tissue Histology is a descriptive science and observation is the key Tissue structure: how cells combine together with extracellular material and each other to form a tissue Cellular structure: cell shape? and how the components inside cells are organized to support that cells specific function Sub-cellular structure: analysis of organelles and inclusions 3  Four Tissue Types Connective Nervous Muscle Epithelial 4  Tissue Components: Cells and ECM ECM consists of many molecules, which are highly organized and form complex structures like collagen fibrils and basement membranes. Mechanical support for the cells, Transport nutrients to the cells, Carry away metabolites and secretory products. Cells not only produce extracellular matrix components but are also influenced by them creating an intense interaction between cells and matrix. 5  Tissue Components: Cells and ECM Endothelium Endothelium Medial layer Medial layer Adventitia layer Adventitia layer 6  Specimen Preparation - Overview Specimen dissection, fixation, preparation, sectioning, staining & microscopy 16 - 48 hours 7  Specimen Preparation The aim of tissue processing is to embed a tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue. FDCES Stages 1. Fixation X 2. Dehydration - Alcohols Remove the water 3. Clearing - Xylene 16 - 48 hours 4. Embedding - Paraffin wax 1 5. Sectioning Long Process 8  Specimen Preparation - Fixation Treatment of a tissue with chemical or physical agents The aim of fixation: To prevent autolysis and bacterial attack. To fix the tissues so they will not change their volume and shape during processing. Stops enzymatic activity. To prepare tissue and leave it in a condition which allows clear staining of sections, stabilises the structure. To leave tissue as close to the living state as possible, and no small molecules should be lost. 9  Specimen Preparation - Fixation Types of fixative: Nothing!! Aldehyde (cross link amine groups of proteins) Ketones i.e. acetone Alcoholic fixative i.e. Methanol (coagulated proteins) Zinc fixative (amino, carboxyl end groups; reversible) Heavy metals i.e. osmium tetroxide Temperature(Freezing) 10  Specimen Preparation - Dehydration To remove fixative and water from the specimen and replace with dehydration fluid. Mostly alcohols - ethanol, methanol etc. To minimize tissue distortion from diffusion currents, specimens are dehydrated in a graded ethanol series from water through: 10 % 20 % 50 % 70 % 95 % 100 % ethanol 11  Specimen Preparation - Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon the following: The type of tissues to be processed, and the type of processing to be undertaken. The processor system to be used. Intended processing conditions such as temperature, vacuum and pressure. Safety factors Cost and convenience Speedy removal of dehydrating agent Ease of removal by molten paraffin wax Minimal tissue damage 12  Specimen Preparation - Clearing Typically used clearing agents: Zylene Toluene Chloroform Benzene Petrol Histo-clear® HISTOCHOICE 13  Specimen Preparation - Embedding Process by which tissues are surrounded by a medium which will provide sufficient external support during sectioning. Embedding medium Paraffin wax (sections ≥ 3 μm) Plastic Resin (very thin sections 0.5 - 1 μm) Polymerizing resin i.e. Epoxy, Aradite Cryo-embed medium (sections ≥ 5 – 100 μm) Agar (sections 100 - 300 μm unfixed, 10 - 20 μm fixed) 14  Specimen Preparation - Embedding Paraffin wax polycrystalline mixture of solid hydrocarbons It is about two thirds the density and slightly more elastic than dried protein. Marketed by its melting points which range from 39°C to 68°C. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: Improve ribboning Increase hardness Decrease melting point Improve adhesion between specimen and wax 15  Specimen Preparation - Embedding The wax must be free of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size. 16  Specimen Preparation - Embedding The wax must be free of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size. 17  Specimen Preparation - Embedding 18  Slide Preparation - Sectioning In order to facilitate handling and allow for staining embedded tissues are sectioned using a microtome. Sections typically taken at 4 - 40 μm for light microscopy Usually Mounted onto glass slides 19  Cryo-embedding & Sectioning Tissue samples snap frozen in LN 2 or CO2 Embedded using a medium which is solid below -10 °C Sectioned using a cryostat at > 6 μm Slides must be stored < -20°C Slides often fixed before staining using alcohol or chloroform or 1:1 mixture. 20  Staining Permits the examination of tissues by light microscopy, tissues and cells are translucent, > 1 stain used. Most stains not compatible with paraffin wax Slides cleared in Xylene Rehydrated through alcohol series Washed in water to remove alcohol Stained Dehydrated Cleared in Xylene Mounted using xylene medium and glass coverslip 21  Staining - Haematoxylin and eosin (H&E) H & E is a charge-based, general purpose stain. Haematoxylin: cationic dye (+ charge) that binds to negatively charged (acidic) structures in the cell. Nucleus = Blue Eosin: anionic dye (- charge) adheres to basic structures in the cell. Amine groups (NH3+) on proteins make cytoplasm = Pink 22  Staining - DAPI Fluorescent stain which binds to A-T rich regions (minor grove) in DNA. DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells. Double stranded DNA, therefore nuclei Blue The first image excitation at 350 nm and collecting reflected light through a 470 nm emission filter, the second image was captured using excitation at 490 nm and collecting light through a 525 nm emission filter. 23  Staining - Alcian Blue Alcian blue is a mucin stain that stains certain types of mucin blue. Cartilage is also stained blue. It can be used with H&E, nuclear fast red, and with van Gieson stains. Acid mucins Blue Proteoglycans Blue Nuclei Red/Black 24  Staining - Oil Red O Oil Red O is used to stain fat in unfixed frozen sections Processing may remove the fact content from cells and tissues Fat Brilliant red Nuclei Blue 25  Staining - Millers Sirius Red Combination stain viewed under Köhler illumination and phase contrast Köhler Elastin Dark purple/ Black Collagen Red/ Pink Phase contrast Collagen fibers are birefringent 26  Staining - Masson’s Trichrome This is often used to stain connective tissue. Nuclei and other basophilic (basic-liking) structures blue cytoplasm, muscle, erythrocytes and keratin bright-red Collagen is stained green or blue Depending on which variant of the technique is used 27  Staining - PAS (Periodic Acid Solution) Basic fuschin that reacts with aldehyde groups. Carbohydrates and carbohydrate rich macromolecules a deep red colour (magenta) Glycoproteins Magenta Nuclei Blue Spleen 28  Staining - Reticulin Gordon and Sweet’s Silver Reduction of silver ions into silver metal and its deposition on the reticulin fibers (mainly collagen type III). Reticulin fibers Black Collagen fibers Brown Nuclei Pink 29  Immunohistochemistry (IHC) IHC is an application of antibodies to tissue preparation for the localization of specific antigens: wide range of specific antibodies highly sensitive detection system Antibody binding is visualized using a marker such as: fluorescent dye enzyme, radioactive element colloidal gold 30  Immunohistochemistry - Direct Method Labeled Antibody Tissue Antigen 31  Immunohistochemistry - Indirect Method Fluorescence Secondary Antibody Fluorescently labeled Primary Antibody Tissue Antigen 32  Immunohistochemistry - Indirect Method Enzymatic Avidin biotinylated Biotinylated complex Secondary Antibody Primary Antibody Enzyme substrate Tissue Antigen 33  Immunohistochemistry - Antigen Retrieval The tissue antigens can be masked as a result of tissue fixation or tissue processing Enzymatic digestion Citric Acid EDTA Heat 34  Immunohistochemistry - Pretreatment Antigen Retrieval Enzymatic digestion Citric Acid EDTA Heat Inhibition of endogenous tissue components 3 % (v/v) H2O2 0.01 % (w/v) avidin Blocking of nonspecific sites 10 % (v/v) normal serum 35  Immunohistochemistry - Controls Positive control tissue known to contain epitope Diluent control buffer only Omission of primary antibody Omission of secondary antibody Isotype antibody identical isotype against a different antigen 36  Immunohistochemistry - Examples Collagen type IV 37 α-Gal  Immunohistochemistry - Fluorescent Examples Smooth muscle cells Human Skin DAPI E-cadherin Actin DAPI MHC 1 38  Immunohistochemistry Advantages 1. High specificity for molecular species 2. Can be used for light, confocal, or electron microscopy Disadvantages 1. Time consuming & expensive 2. Fixation can interfere with Ab binding 3. Reproducibility - false positives - cross reactivity 4. Difficult to get Abs to small molecules 5. Qualitative 39  Microscopy - Bright Field Light Microscope Kohler Illumination Objectives and eyepieces focus an image of the illuminated specimen in the eye. Light is focused onto the specimen via the condenser. (Köhler Illumination) 40  Microscopy - Phase contrast Phase contrast microscopy allows the viewing of unstained specimens by using the light phase amplitude differences within microscopic objects 41  Microscopy - DIC Nomarski or differential interference contrast (DIC) microscopy Interference. Uses polarized light in combination with a variable prism to alter the interference patterns Images can be seen in striking colour (optical contrast) with a 3-dimensional shadowed. Investigation of living cells, non-invasive and real-time, optical sectioning possibilities allow the movement of tiny organelles to be followed. 42  Microscopy - Fluorescence Light at specific wavelengths is focused on the specimen via an objective which excites a fluorophore. Emitted light is captured at a different wavelength 43  Microscopy - Confocal Uses a laser to excite fluorescent dye in tissue or cell one spot at a time Precisely positioned pinholes allow only in-focus light to pass through Images in XYZ planes Computer takes all images and creates a 3D reconstruction Advantages Excellent resolution in thick samples Greater number of flurophore as specific wavelengths used to illuminate samples Collects light from a single focal plane Disadvantages Photo bleaching and phototoxicity Increased sensitivity to noise Technical method - labour intensive 44  Histology - Interpretation 45  Enzyme Linked Immunosorbent Assay (ELISA) Different Types a) Sandwich b) Indirect c) Competitive Can be used to detect both antibody and antigen Very sensitive, pg.mL-1 - 2-3 S.D above background signal Relies on monoclonal antibodies An enzyme’s activity is used as a “reporter The enzymatic reaction will produce a colored species Standard curve used to determine unknown sample 46  Enzyme Linked Immunosorbent Assay (ELISA) 47  Additional Resources www.Histology.leeds.ac.uk Theory and Practice of Histological Techniques John D Bancroft Wheater's Functional Histology: A Text and Colour Atlas Barbara Young http://www.dako.com/uk/index/knowledgecenter.htm http://docs.abcam.com/pdf/misc/abcam-protocols-book-2010.pdf http://www.abcam.com/index.html?pageconfig=popular_protocols www.ihcworld.com Microscopy http://www.olympusmicro.com http://zeiss-campus.magnet.fsu.edu/articles/basics/index.html 48 49