L2 and L3 - TISSUE PREP & MICROSCOPY.pdf

ITM 101 | HISTOLOGY LESSON # 2 - Fixation 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ 6. Sectioning - Uses a microtome — used OBJECTIVES...

ITM 101 | HISTOLOGY LESSON # 2 - Fixation 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ 6. Sectioning - Uses a microtome — used OBJECTIVES for sectioning Understand the basic concepts of tissue fixation, paraffin-embedded tissues dehydration, embedding, sectioning, staining, and for light microscopy. The mounting of slides for histological examination, tissue block passes over immunofluorescent staining and electron the steel knife edge and a microscopy section is cut at a Recognize, identify and describe the characteristic thickness equal to the structures of cells, tissues and organ systems of distance the block the body at the light microscope histologic level, advanced. and for selected tissues, at the electron microscopic ultrastructural level TISSUE PREPARATION 7. Staining 1. Biopsy 8. Completion of Slide preparation 2. Fixation - can be alcohol based (e.g. formalin) and non-alcohol based FIXATION preserve tissue by preventing autolysis by cellular enzymes and decomposition by bacteria Harden tissue — to allow think sectioning Devitalize or inactivate infectious agents Stabilize tissue components Enhance avidity for dyes Adequate Fixation depends upon: 3. Trimming ○ Sufficient volume At least 15-20 times greater than tissue volume ○ Access of fixative to tissue 6-8 hours To allow fixative to penetrate slowly Techniques: use of gauze 4. Tissue processing pads, pinning on paraffin blocks Size Large specimens must be thinly sliced Temperature Room temp Formalin fixatives ○ 10% phosphate-buffered formalin ○ It fixes most tissues well and is compatible with 5. Embedding most histologic stains ○ Fixation occurs due to cross linking of proteins ○ Overfixation can diminish immunoreactivity ○ Formalin penetrates tissue at about 0.4cm each 24hrs Non-formalin fixatives ○ Many are alcohol based ○ May be used to avoid formaldehyde ○ Nuclear and cytologic detail may not be as good as with formalin 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 1 ITM 101 | HISTOLOGY LESSON # 2 - Fixation 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ Non-Formalin Fixatives Description retrieval: Protease-Induced Epitope Retrieval (PIER) and Heat-Induced Epitope Retrieval can be used in any tissue but (HIER) Bouin’s solution causes brittleness if ever fixed B-plus (clear) for lymph nodes, spleen Dewaxing/ Removing the wax Zenker’s acetic fixative for bone marrow ○ Following the preparation of the paraffin slow fixative but excellent section, all the elements are infiltrated with and Glutaraldehyde preservation. Tissues have to surrounded by paraffin wax which is be minced into small cubes hydrophobic and impervious to aqueous reagents. The majority of cell and tissue indicated for synovial components have no natural color and are not specimens since urate crystals visible. The first step in performing an H&E will be dissolved by water Alcohol stain is to dissolve all the wax away with xylene containing fixatives such as (a hydrocarbon solvent). formalin. Ethanol and methanol Hydrate the Section will harden the tissue ○ After thorough de-waxing, the slide is passed through several changes of alcohol to remove DEHYDRATION the xylene, then thoroughly rinsed in water. The The water in the tissue is replaced by alcohol — The section is now hydrated so that aqueous tissue is transferred through a series of increasingly reagents will readily penetrate the cells and concentrated alcohol solutions, ending in 100%, which tissue elements. removes all water. Complete the Nuclear Stain by “Blueing” Nonaqueous embedding media (such as paraffin) cannot ○ After rinsing in tap water, the section is “blued” penetrate tissue containing water by treatment with a weakly alkaline solution. This step converts the hematoxylin to a dark CLEARING blue color. The section can now be rinsed and checked to see if the nuclei are properly The alcohol is replaced by a clearing agent that makes stained, showing adequate contrast and to the tissue receptive to infiltration by the embedding assess the level of background stain. medium Remove Excess Background Stain (Differentiate) ○ On most occasions when Harris hematoxylin is INFILTRATION employed, a differentiation (destaining) step is The xylene is replaced by paraffin or another embedding required to remove nonspecific background medium. The paraffin stiffens the tissue, and this allows staining and to improve contrast. A weak acid very thin sections (only a few microns in thickness) to be alcohol is used. After this treatment, blueing cute with a microtome and thorough rinsing is again required. Staining methods that include a destaining or EMBEDDING differentiation step are referred to as At the end of processing step, the cassettes containing “regressive” stains. tissue are immersed in paraffin and allowed to harden Dye SECTIONING “CUTTING” The “block” (the tissue embedded in paraffin attached to 1. Hematoxylin: Basic, used to illustrate the bottom of the cassette) is mounted on a microtome positively charged nuclear detail in cells and a four micron section is cut from the surface of the dye Depth of coloration is block not only related to the The cut tissue is floated in a water bath – lets tissues amount of DNA in the relax and smoothen before being mounted on slide as nuclei but also to the well as makes paraffin stick to the slide. This ensures length of time the removal of wrinkles and folds before sections are placed sample spends in on the slide. hematoxylin. STAINING extracted from the tree H&E (Hematoxylin & Eosin) Haematoxylum ○ Follow procedure: campechianum. 1. Dewaxing Oxidation of the 2. Dehydration hematoxylin produces 3. Hematoxylin hematein, which is the 4. Differentiation actual dye used in an 5. Bluing H&E stain 6. Eosin Addition of the 7. Dehydration mordant improves the 8. Clearing ability of the hematein 9. Cover slipping to attach to the anionic (negatively charged) components of the Antigen retrieval tissues. ○ There are two main methods of antigen 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 2  ITM 101 | HISTOLOGY LESSON # 2 - Fixation 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ Overstaining can make non-cancer cells look like cancer cells 2. Eosin: Acidic, the most commonly negatively charged used counterstain that dye distinguishes between the cytoplasm and nuclei of cells. It is typically pink, with different shades of pink for different types of connective tissue fibers. Eosin Y is the most commonly used form Cells lining BV “endothelium” of eosin and may be used in both water and alcohol. 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 3 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ SIMPLE COLUMNAR EPITHELIUM OUTLINE The cells have rectangular outlines with their long axis Basic Tissues perpendicular to the basal lamina. ○ Epithelium Found in the digestive tract from the cardia of the ○ Connective tissue stomach to the anus, ○ Blood Ciliated simple columnar is found in the uterus, and ○ Muscle oviducts. ○ Nervous Tissue Involving tissues that are mainly for storage Classification of Epithelia ○ Simple epithelium ○ Stratified Epithelium 1. Squamous 2. Cuboidal 3. Columnar 4. Ciliated Glands & Exocrine ○ Exocrine Merocrine STRATIFIED SQUAMOUS EPITHELIUM Apocrine The cells vary in shape from base to the free surface. Holocrine The cells on the basal lamina have rounded or beveled ○ Endocrine upper ends. Those above this layer are irregularly polyhedral , becoming increasingly flattened toward the surface , and in the superficial layers they are thin EPITHELIUM squamous cells. SIMPLE SQUAMOUS EPITHELIUM When stained with nitrate, a tile-like pattern of closely adherent cells with polygonal outline Found in the pulmonary alveoli, parietal layer of Bowman’s capsule, thin segments of loop of henle, rete testis, tympanic membrane, endothelium lining the blood vessels and lymph vessels. – single layered tissues involved in exchange of particles (e.g. exchange of gasses, filtration, reabsorption) STRATIFIED COLUMNAR EPITHELIUM The superficial cells are columnar and the basal cells are cuboidal with a rounded upper end. Found in the fornix of the conjunctiva, cavernous urethra, and pharynx SIMPLE CUBOIDAL EPITHELIUM The epithelium appears as a row of square or rectangular profiles. Found in the follicles of a thyroid, mammary ducts, PSEUDOSTRATIFIED COLUMNAR EPITHELIUM choroid plexus, capsules of the lens – tissues mainly All cells are in contact with the basal lamina but only a involving secretion functions portion of them reach the surface. Cells are variable in shape. Some are wider in the base. Found in the trachea, and primary bronchi of the respiratory tract and auditory tube. 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 4 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ TRANSITIONAL EPITHELIUM CARTILAGE TISSUE intermediate between columnar and stratified squamous Found in the tracheal rings,nose, larynx, joint surfaces, epithelium ends of the ribs that connect to the sternum Mainly occurs in the urinary bladder Semi Translucent tissue with bluish gray color. Chondrosarcoma is a type of bone cancer that develops in cartilage cells. Cartilage is the specialized, gristly connective tissue that is present in adults and the tissue from which most bones develop. Cartilage plays an important role in the growth process. BLOOD Elastic Cartilage ○ Found in the external ear, walls of the auditory canal, and Eustachian canal, epiglottis, corniculate and cuneiform cartilage of the larynx ○ Greater opacity, yellowish color and greater flexibility. ○ Matrix is less abundant – covered by elastic fibers Ellergy Red blood cells (erythrocytes) ○ Shape: biconcave disc Fibrocartilage ○ Size: approximately 7.5 to 8.7 μm in diameter ○ Found in insertion of ligaments and tendons into and 1.7 to 2.2 μm in thickness bone. ○ They stay for 120 days in the bloodstream ○ In place of fusiform fibroblasts, chondrocytes ADIPOSE TISSUE surrounded by a small amount of cartilage matrix are aligned in rows between coarse parallel bundles of type 1 collagen fibers. Abundant in cytoplasm due to lipid droplets Peripheral nucleus Liposarcoma is a rare tumor derived from fat tissue that occurs in the “soft tissues” of the body (soft tissue sarcoma) ○ Benign in younger compared to older people 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 5 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ BONE TISSUE Cortical bone Haversian canal – where nutrients circulate Bone formation a. Intramembranous ossification – directly from mesenchyme (embryonic connective tissue where all bones are derived) b. Endochondral ossification – from cartilage derived from mesenchyme Special cells ○ Osteoclast – bone resorption “CRUSHING” – cells that degrade bone to initiate normal bone remodeling and mediate bone loss in pathologic conditions by increasing their resorptive activity ○ Osteoblast - bone maturation “BUILDING” – synthesize and secrete bone matrix and participate in the mineralization of bone to regulate the balance of calcium and phosphate ions in developing bone MUSCULAR TISSUE The units of organization of skeletal muscle are the muscle fibers, long cylindrical multinucleated cells. Striated; peripheral nucleus The light bands are called I bands and contain only thin filaments. The dark bands are called A bands and contain thick and thin filaments, with the thick filaments running the entire length of the A band. Sliding Filament Theory 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 6 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ NERVOUS TISSUE GLANDS & EXCRETION Endocrine glands Exocrine glands Fact: Pancreas is both endo & exocrine ENDOCRINE secrete products directly into the bloodstream Example: Pancreas – secretes insulin & glucagon in the islets of Langerhans LYMPH NODES EXOCRINE secrete substances into a ductal system to an epithelial surface 3 Mechanism of Product Release 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 7 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ A. Merocrine - Release of product with the cell membrane remaining intact. - secretory vesicles fuse with the apical plasma membrane to release their products into a duct (e.g. secretion of saliva by salivary glands). B. Apocrine - Involves constriction and pinching off of a portion of the apical cytoplasm containing the secretory granules. - e.g. secretion of lipid droplets in the mammary gland - Smallest unit: Terminal duct-lobular unit INTESTINE C. Holocrine They are of two types namely, - Consists of bulk release of whole cells or crypts of Lieberkuhn and discharge of the cytoplasm into the excretory ducts of the gland. Brunner's glands. They - Classic example: Sebaceous gland where the secrete digestive enzymes cells break down with an outpouring of and mucus. cytoplasm and accumulated lipid rich secretion. - E.g. sperm Example: Pancreas – responsible for secretion of digestive enzymes, ions and water into the duodenum of the gastrointestinal tract RESPIRATORY STOMACH The stomach holds multiple types of exocrine glands that include: cardiac, gastric and pyloric, named after the region in which they are found. These glands produce the Submucosal glands contribute to airway surface liquid (ASL), a digestive enzymes and mucous film that protects all airway surfaces. Glandular mucus comprises secretions of the stomach. electrolytes, water, the gel-forming mucin MUC5B, and hundreds of different proteins with diverse protective functions. 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 8 ITM 101 | HISTOLOGY LESSON # 3 - Microscopy 1st YEAR | A.Y. 2023-2024 | AUGUST 23, 2023 DR. MARQUEZ LIVER Largest gland in the body Hepatocytes are arranged into cords Separated by vascular sinusoids the cord is one cell thick As an endocrine gland, it produces and secretes plasma proteins, clotting factors, and insulin-like growth factors into the bloodstream. On the other hand, as an exocrine gland, the liver secretes bile into the digestive system for the chemical digestion of lipids References Lester. Manual of surgical pathology 3rd J., L. L. M. (2014). Lippincott’s Pocket Histology. Wolters Kluwer Health/Lippincott Williams & Wilkins. Bloom, W., & Fawcett, D. W. (1994). A textbook of histology. Chapman & Hall. MESCHER, A. (2023). Junqueira’s basic histology: Text and Atlas. MCGRAW-HILL EDUCATION. 101 INTRODUCTION TO MEDICINE | HISTOLOGY AYURA 2027 9