Topic 1: Introduction to Histology Animal Histology BIOL 241 Topic 1: Introduction to Histology Dr. Issa Al-Amri Department of Biological Sciences & Chemistry College of Arts & Sciences

Histological Techniques Methodology (Histological Techniques): Type of sample preparation methods: Preparation methods for light microscopy To prepare tissue for LM examination, fixed tissues used. Samples of fixed tissues are used in the form of: Sections on glass slides. Smears on glass slides for blood, mucus, urine, and bone marrow. Spreads for loose (areolar) connective tissue. Teased samples for muscle tissue (individual fibres).

Histological Techniques Tissue processing: Removal of specimen: tissue specimen dissected from the animal and placed in fixative. Fixation: fixing fluids (buffered 10% formaldehyde) preserve structures (cross-link proteins) and inhibit autolysis (digestion by enzymes) and bacterial growth. Dehydration & clearing: dehydration is removal of water from tissue and replace it with graded conc. of ethanol. Clearing is replacing ethanol with solvent (xylene) that mix with paraffin wax. Process carried in tissue processor. Infiltration: gradual replacement of xylene with wax (60C) Embedding: tissue embedded in paraffin provides rigid support to tissue blocks to be cut into thin sections. Embedding station used for tissue orientation and wax.

Histological Techniques Fixation of animals (formalin)

Histological Techniques Formalin fixed brain

Histological Techniques Tissue dissected and placed in cassette for processing

Histological Techniques Automatic Tissue Processer

Histological Techniques Tissue Embedding Station

Histological Techniques Tissue embedding process – processed tissue removed from cassette

Histological Techniques Tissue embedding process – tissue oriented in a mould

Histological Techniques Tissue embedding process – paraffin added to fill cassette and mould

Histological Techniques Tissue embedding process – wax solidification in cold plate

Histological Techniques Tissue Blocks ready for sectioning

Histological Techniques Sectioning (microtomy): The tissue block then cut into thin sections 1 - 10 µm with a rotary microtome. Sections then mounted on glass slides and stained. Frozen sections obtained after tissue fixation by freezing, then cut into sections by cryostat (freezing microtome), mounted on glass slides and stained. This method used in hospitals for quick diagnosis during surgery and to study the enzymes by histochemical methods.

Histological Techniques Rotary Microtome

Histological Techniques Sectioning processes

Histological Techniques Ribbon of Paraffin Sections into water bath

Histological Techniques B Ribbon of Paraffin Sections in water bath. A. Sections with wrinkles. B. Sections stretched

Histological Techniques Paraffin Sections picked up in glass slide

Histological Techniques Sections in glass slide left to dry in hot plate

Histological Techniques Histological Staining To stain tissue and make some parts appears darker than others is by use of dyes which stain tissue components selectively. Dyes are either acidic or basic. Tissue structures stained with basic dyes such as haematoxylin, methylene blue, and toluidine blue, are said to be basophilic. Tissue structures stained with acidic dyes such as eosin, is said to be acidophilic.

Histological Techniques Histological Stains Routine H&E stain: Routine staining of tissue usually employs Haematoxylin (H) and Eosin (E) stains to stain general structures. Haematoxylin reacts with nucleic acids and acidic groups of proteins and stain them blue. The nucleic acids are concentrated in the nucleus and rough endoplasmic reticulum (RER). Eosin, stains the cytoplasm of most cells red or pink. The cytoplasm may be basophilic, acidophilic or neutrophilic.

Histological Techniques - Harris’s H&E Staining Procedure: Sections (in slides) deparaffinised in xylene. Sections then hydrated through graded ethanol concentration, and then washed in running tap water. Slides then stained with Harris’s haematoxylin, then washed in running tap water. Slides then differentiated in 1% acid alcohol, and rinsed in running tap water. Slides taken into blueing solution (weak ammonia), then rinsed in running tap water. Slides stained in 1% aqueous eosin Y, then rinsed in tap water. Slides dehydrated through graded alcohol concentration, and cleared through a series of xylene changes. Slides then mounted in D.P.X. , covered with coverslip.

Histological Techniques Manual staining

Histological Techniques Automatic staining

Sections stained with H & E stain, mounted with D.P.X , covered Histological Techniques Sections stained with H & E stain, mounted with D.P.X , covered with coverslips

Histological Techniques Kidney glomerulus stained with H & E stain

Digital light microscopy: imaging using digital camera, computer with camera program and a monitor

Histological Techniques Histological Staining Special stains: Used to stain special structures such as glycogen granules and fibrils. Counter stain (e.g. Mayer’s haematoxylin) is then used to reveal nuclei and the background of special structures. Trichrome stains such as Mallory and Masson`s stains applied to differentiate between collagen and smooth muscle fibres.

Histological Techniques Histochemical and cytochemical staining: Methods used to localize substances in tissue sections based on specific chemical reactions between macromolecules and production of insoluble coloured substances seen under microscope. Examples of studied substances: Ions: such as calcium, iron, and phosphate. b. Nucleic acids: DNA and RNA studied by use of basic dyes.

Histological Techniques Proteins: study of enzyme activity in tissue by localising the action of enzyme with its substrate by producing coloured insoluble substance that can be seen under the microscope. The most common enzymes studied: Phosphatase: acid phosphatase (lysosomes in macrophages) and alkaline phosphatase (liver). Dehydrogenase: succenic dehydrogenase of mitochondria. Peroxidase: present in liver and kidney cells; it promotes oxidation of its substrates.

Histological Techniques d. Polysaccharides and oligosaccariades: carbohydrates present either free (glycogen) or as combined with lipids as glycolipids or with proteins as glycoproteins. Carbohydrate part can be demonstrated by use of PAS reaction. PAS (Periodic Acid Schiff reagent): method used to stain glycosamino-glycan [GAG], glycoproteins, and glycogen which then counterstained by hematoxylin. e. Lipids: lipids dissolve in routine preparations, best to use dyes that dissolve in lipids. Frozen sections dipped into alcohol saturated with Sudan IV or Sudan black which stains lipid droplets red or black respectively.

Histological Techniques Common Stains in Light Microscopy: H & E: routine; stains nuclei blue and the cytoplasm pink. Azocarmine: used as a counter stain. Stains glycogen and mucigen. Mallory-Azan: stains nuclei red and the cytoplasm orange or blue. Masson`s trichrome: stains nuclei black or dark blue, cytoplasm stains red, reticular and collagen fibres stain blue. Periodic acid-Schiff`s and hematoxylene (PASH): stains glycogen, mucin and carbohydrate containing tissue red or pink and the nuclei blue. Silver stain: silver nitrate used to stain reticular fibers, neurofibrils and granules in enteroendocrine cells. Orcein stain: for elastic fibres.

Histological Techniques Thionin stain: stain Nissl`s granules blue in nerve cells. Toluidine blue: stains granules in mast cells reddish-purple, while nuclei stained deep blue. Trypan blue: vital stain living cells in vivo, or supra vital stain living cells in vitro. In vivo, stain injected into living animals to demonstrate phagocytic cells which ingest stain granules. Verhoff`s stain: stains elastic fibres black. Osmium tetroxide: stain lipid contents of cells black colour e.g. myelin sheath of nerve fibres. Sudan III: stains lipids with orange colour e.g. fat droplets in adipocytes.

Histological Techniques

Histological Techniques PAS-Fast Green. Uterine tube. cytoplasm of nonciliated cells (NC) contains secretory granules (SG).

Histological Techniques Masson’s Trichrome stain showing glomerulus with slight mesangial prominance.

Histological Techniques Masson’s Trichrome stain of muscular artery : Collagen and nerve cells stained blue. Muscle, elastic fibres and blood stained red. Cytoplasm of cells stained red. Nuclei stained purple.

Histological Techniques Mallory-Azan stain of fibres

Histological Techniques Immunohistochemistry: Highly specific reaction between an antigen (foreign protein) and its antibody (immunoglobulin). Tissue sections incubated with antibodies to their contents of proteins so each antibody binds specifically to its protein (antigen) denoting the protein location in the cell that can be seen under the microscope. Polyclonal antibodies: antibodies produced and collected contain mixture of several groups of antibodies for different parts of proteins (antigens). Monoclonal antibodies: antibodies for different parts of proteins are collected separately.

Histological Techniques Localization of IgG4 (red) in mouse Pancreas

Histological Techniques Alzheimer :Tau in Neurofibrillary tangles in human brain

stained positive in nuclei and cytoplasm (arrow). Histological Techniques Immunolocalization of previtellogenic follicle PRs. All granulosa cells stained positive in nuclei and cytoplasm (arrow).