Introduction to Histology Light and Electron Microscopes Tissue Preparation Prof. Abdulameer Al-Nuaimi E-mail: a.al-nuaimi@sheffield.ac.uk E. mail: abdulameerh@yahoo.com

Histology : Is a branch of anatomy that deals with the minute structure of animal and plant tissues. The fundamental aim of histology is to determine how tissues and organs are organized at all structural levels.  It is commonly performed by examining cells and tissues under light microscope or studying the ultrastructure components of cells under Electron microscope. Steps taken to prepare tissue for histological study Specimen is usually taken from a selected organ, Fixed, Embedded, Cut into a thin cross section with a microtome Mounted on a microscope slide, Stained Examined under the microscope

Animals Animal rights is the idea that some or all nonhuman animals are entitled to the possession of their own lives, and that their most basic interests (such as an interest in not suffering) should be afforded the same consideration as the similar interests of human beings Experimental animals should be respected

Dissecting Board Made of thick high-density polyethylene to retain shape without bending or swelling, and can be sterilized All corners have rubber feet for stability

fixation

Tissue processing 1- Tissue Fixation Objective of tissue fixation is to preserve cells and tissue components and keep them as close to normal as possible and allow for the preparation of thin, stained sections. Fixation is usually the first step in a process to prepare a sample of biological material for microscopy or other analysis.

Fixatives work in the following way. 1-Fixative usually acts to disable intrinsic biomolecules—particularly proteolytic enzymes—which otherwise digest or damage the sample. 2-Fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample 3-fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This helps in preserving the morphology (shape and structure) of the sample as it is processed for further analysis

Choice of fixative and fixation protocol It depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used

Types of fixation Heat fixation: preserves overall morphology but not internal structures. Freezing: Used to get rapid results but does not give fine details Chemical fixation In this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble

Target Fixative of Choice Fixative to Avoid Proteins Neutral Buffered Formalin, Paraformaldehyde Osmium Tetroxide Enzymes Frozen Sections Chemical Fixatives Lipids Frozen Sections*, Glutaraldehyde/Osmium Tetroxide Alcoholic fixatives, Neutral Buffered Formalin Nucleic Acids Alcoholic fixatives, HOPE Aldehyde fixatives Mucopolysaccharides Chemical fixatives Biogenic Amines Bouin Solution , Neutral Buffered Formalin Glycogen Alcoholic based fixatives

Factors Affecting Fixation 1-pH of the fixative Should be kept in the physiological range, between pH 4 to 9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4 2-Osmolarity of the fixative: Try to avoid Hypertonic solutions :give rise to cell shrinkage. Hypotonic solutions :result in cell swelling and poor fixation. 10% neutral buffer formalin fixative (4% formaldehyde in phosphate buffered saline), is a very hypertonic solution, yet it has worked well as a general tissue fixation condition 3-Size of the Specimen : Ideal thickness is 1-4mm 4-Volume of the Fixative : At least 15-20 times greater than tissue volume

5-Temperature: High temperature increases the speed of fixation 5-Temperature: High temperature increases the speed of fixation. However, care is required to avoid cooking the specimen. Fixation is routinely carried out at room temperature. 6-Duration: As a general rule 1hr per 1mm Fixation is a chemical process, and time must be allowed for the process to complete. Although "over fixation" can be harmful, under-fixation has been appreciated as a significant problem and may be responsible for inappropriate results for some assays

2- Embedding Embedding means blocking tissue within hard medium (paraffin or resines) The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Embedding material For light microscopy, paraffin wax is most frequently used. For electron microscopy, resins are the most commonly used. During embedding, the tissue samples are placed into molds along with liquid embedding material which is then hardened. Formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature,

Automatic Tissue Processor

Tissue Embedding Machine

3- Tissue Sectioning (Cutting) For light microscopy, a steel knife mounted in a microtome is used to cut 3 -5 µm -thick tissue sections which are mounted on a glass microscope slide. 1mm = 1000 µm For transmission electron microscopy, a diamond knife mounted in an ultramicrotome is used to cut 0.1-0.5 µm thick tissue sections which are mounted on a 3-mm-diameter copper grid. Then the mounted sections are treated with the appropriate stain. Frozen tissue embedded in a freezing medium and cut on a microtome in a cooled machine called a cryostat. It cuts 1-10 µm thick sections.

Paraffin sectioning Microtome

4- Mounting the sections on a microscope slide Sections are mounted onto gelatin-coated histological slides. Slides are pre-coated with gelatin to enhance adhesion of the tissue. Sections are floated in a 56 °C water bath and then mounted on the slides Mounted sections are dried on a hot plate at 56 °C and then Kept overnight at room temperature. Slides with paraffin-embedded sections can be stored either at room temperature or at 2-8 °C for several years in slide storage boxes.

5- Staining Biological tissue has little contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. Hematoxylin and Eosin (H&E stain) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus. Eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to add contrast to tissue in the electron microscope. Histochemistry: shows chemical reactions between laboratory chemicals and components within tissue.

Histochemistry stains PAS Stain PAS stains carbohydrates and carbohydrate rich macromolecules a deep red colour Masson's trichrome stain. This is often used to stain connective tissue. Collagen is stained green or blue Van Gieson stain This stains collagen red, nuclei blue, and erythrocytes and cytoplasm yellow.

immunohistochemistry. Antibodies are used to specifically visualize proteins, carbohydrates, and lipids. this photo shows some cells that have been immunofluorescently stained for the protein actin.

History of staining In the 19th century, histology was an academic discipline in its own right. The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y. Cajal. They had given interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible Santiago Ramon y Cajal

Normal skin

Normal Skin

Normal Lung

Light Microscope

Light Microscope

Electron Microscope

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