Histology Lab (001) PDF - University of Northern Philippines

(001) HISTOLOGY LAB DR. CABUENA | 09/14/2020 -better used together but can be also used separately, OUTLINE...

(001) HISTOLOGY LAB DR. CABUENA | 09/14/2020 -better used together but can be also used separately, OUTLINE however it will limit the cellular structures being I. THE MICROSCOPE shown A. COMPONENTS OF THE LIGHT MICROSCOPE II. HISTOLOGICAL STAINS - The cell membrane is the cytoplasmic component A. MOST COMMON TYPES OF HISTOLOGICAL STAIN that is not clearly distinguishable by Hematoxylin and B. OTHER STAIN Eosin but the cytoplasm appears pinkish or reddish. III. STAINING PATTERN OF THE NUCLEI The nucleus appears intensely blue or purple in color. IV. ADDITIONAL HISTOLOGICAL SAMPLES A. MOST COMMON TYPES OF HISTOLOGICAL STAINS/ CYTOPLASMIC STAINS I. THE MICROSCOPE 1. Basic dyes- have a net positive charge and bind to components of  Microscope -instrument used to see objects that are too small cells and tissues that are negatively charged. to be seen by the naked eye -Basophilic- tissue components that stain with basic  Light Microscope- forms a magnified image focusing light with dyes. glass lenses in a thin tissue section  Negatively charged cells I.e: -Phosphate groups of nucleic acids (DNA A. COMPONENTS OF A LIGHT MICROSCOPE and RNA) -Sulfate groups of some polysaccharides 1. Condenser Lens- focuses the light from the illumination (glycosaminoglycans) and some proteins source onto the specimen. (mucus) 2. Objective lens- collects light that passes through the Example: Toluidine Blue, Alcian Blue, Methylene Blue specimen, enlarges and projects an image onto the eyepiece. Hematoxylin behaves like a basic dye, staining basophilic 3. Eyepiece lens- further magnifies images and project onto the tissue components; because of the acid composition of the main retina tissue components (i.e., DNA, RNA, Glycosaminoglycans) 4. Bright-field illumination- is used with specimens stained with 2. Acidic dyes- have a net negative charge and bind to components colored dyes. of cells and tissues that are positively charge -it has a resolution of ~0.25 µm (or ~1000x - stain the acidophilic components of tissues such as greater than the unaided eye) mitochondira, secretory grannules and collagen - Example: Eosin, Orange G and Acid Fuchsin -Acidophilic- tissue components that stain with acid dyes.  Positively charged cells I.e: -Ionized amino groups in proteins (side chains of lysine and arginine) Figure 1.1 Parts of a microscope Figure 1.2 LIght Microscope magnification II. HISTOLOGICAL STAINS  Hematoxylin and Eosin (H&E stain)- Most commonly used stains in histology. -works well with a variety of fixatives and stains a Figure 2. Basophilic and Acidophilic structures. broad range of cytoplasmic, nuclear and extracellular matrix features. PREPARED AND EDITED BY: LIMBAUAN, J., LIVED, R., LOCQUIAO, C., LOPEZ, F., PADOLINA, J., PALAGANAS, B., PANG-AG, L.  (001) HISTOLOGY LAB DR. CABUENA | 09/14/2020  Hematoxylin - positively charged, blue dye complex that stains - Considered as a counterstain basophilic structures. - Single-dye application to distinguish additional features of a tissue - Trichrome stains (Mason trichrome) – for more complex procedure; allow greater distinctions among various A. extracellular tissue components Another example : B.  Trachea- also shows example of both basophilia and C. acidophilia  Acidophilia (or eosinophilia) can be seen within cells such as: -Red blood cells and duct cells Figure 3. Pancreatic cells stained with hematoxylin. Where (A) is the nucleus (blue), (B) is the duct and © is the -Major extracellular protein cytoplasm (lightly stained). -Connective tissue protein collagen  Eosin- negatively charged, pink dye that stains acidophilic (or  Basophilia can be seen within and outside cells. eosinophilic) structures. Hematoxylin stains: -the range of staining intensifies of structures is much -DNA and RNA within cells less than hematoxylin. -Negatively charged molecules -granules are seen in the cytoplasm of most cells -I.e Sulphated gycoproteins in mucus - Sulphated polysaccharides found in the extracellular matrix of cartilage A. A. B. Figure 4. Pancreatic cells stained with Eosin. Where (A) is the lightly stained nucleus while (B) is the intensely stained cytoplasm. B. Figure 6. Trachea. (A) the acidophilic side, (B) the basophilic A. side B. OTHER STAINS B.  Toluidine blue- basic dye that binds nucleic acids, preferentially stains RNA Orthochromatic- when it stains structures blue. Figure 5. Pancreatic cells stained with both Hematoxylin and Eosin.  Periodic acid-Schiff (PAS) reaction – utilizes the hexose rings of Where nuclei (A) is stained blue, while the cytoplasm (B) is stained pink. polysaccharides and other carbohydrate-rich tissue structures and stains such as distinctly purple or magenta Note: Hydrophobic structures (such as those rich in fats) do not stain well o Feulgen reaction – modified procedure of PAS; DNA of with H&E cell nuclei can be specifically stained PREPARED AND EDITED BY: LIMBAUAN, J., LIVED, R., LOCQUIAO, C., LOPEZ, F., PADOLINA, J., PALAGANAS, B., PANG-AG, L.  (001) HISTOLOGY LAB DR. CABUENA | 09/14/2020 o Basophilic or PAS-positive material – identified by enzyme digestion, pretreatment of tissue  Sudan Black – useful in diagnosis of metabolic diseases that involve intracellular accumulations of cholesterol , phospholipids  Hepatocytes or glycolipids, or glycolipids. -cytoplasm contains the most intense staining  Metal impregnation techniques – using of silver salts solution to visual certain ECM fibers and specific cellular elements in nervous tissue. A.  Pararosaniline-Toluidine Blue (PT) – stains chromatid shades of purple and cytoplasm and collagen a lighter violet; penetrate plastic sections more readily than H&E and used primarily with B. Acrylic resin-embedded sections to provide better detail of cell and tissue structures.  Picro-Sirius-Hematoxylin (PSH) – Sirius red in a solution of picrid acid stains collagen red and cytoplasm a lighter violet or pink, with nuclei purple if first stained with picro-sirius red. Figure 8. Liver tissue. (A) Hepatocytes and (B) Chromophilic substances  Wright-Giemsa Stain – used to fixed cells of blood and bone marrow smears to demonstrate types of blood cells. o Granules in leukocytes are seen to have differential  Pancreas affinity for the stain components -most contains acinar cells o Nuclei stain purple and erythrocytes stain uniformly -the apical cytoplasm near the lumen contains unstained pink or pinkish orange secretion granules while the basal portion contains a nucleus with a prominent nucleolus -the presence of chromophil substance in () suggests that acinar cells synthesizes large amounts of protein  Purkinje Cells -found in the interface between the two layers of the >Acinar cells- polarized cells clustered around a central cerebellum. lumen which is in the entrance of a duct. -Note at the figure below that the nucleolus is stained A. blue, meaning it is chromophilic. > Nissl/Chromophil substances - contains negatively charged C. B. RNA. -found in free ribosomes and ribosomes bound in the ER. -Appearance of these substances suggests D. that the cell is involved in large amounts of protein production. A. B. Figure 9. Pancreas stained with Toluidine. The figure shows clear C (B) apical cytoplasm and the darkly stained (A) basal portion, as well as the (C) chromophilic and the (D) acinar cells D. III. STAINING PATTERN OF NUCLEI 2 types of staining pattern: 1. Heterochromatin- tightly packed form of DNA. -Heterochromatic nuclei stain uniformly Figure 7. Layer of purkinje cells between the two layers of and intensely with hematoxylin the cerebellum. (AD) Nucleus and (BC) purkinje cells PREPARED AND EDITED BY: LIMBAUAN, J., LIVED, R., LOCQUIAO, C., LOPEZ, F., PADOLINA, J., PALAGANAS, B., PANG-AG, L.  (001) HISTOLOGY LAB DR. CABUENA | 09/14/2020 -indicative of a cell with low transcriptional activity. 2. Euchromatin- is a lightly packed form of DNA  Motor Neurons -Euchromatic nuclei show random clumps of hematoxylin staining mostly at the periphery of the nucleus and the nucleolus. A. -indicative of cells with high transcriptional activity.  Lymph Nodes B. - contains many lymphocytes that surrounds the capillary. C. Lymphocytes- 7-10 µm in diameter and has D. heterochromatic nuclei and very thin pale cytoplasm. -75% of the volume of the cells is nucleus. Figure 12. Motor Neurons.(A) RBC, (B) the nucleus, ©the nucleolus and (D) cytoplasm ***RBC-7.5µm in diameter and can be used to estimate the other sizes of other structures. A. Note: Nuclei occurs in all cells except in RBC and Platelets IV. ADDITIONAL HISTOLOGICAL SAMPLES B.  Golgi Apparatus (Epididymis) Figure 10. Lymph nodes. (A.)The nucleus is an example of a -composed of two layers of cells facing cross sections heterochromatic nuclei which surrounds the (B) of the duct separated by a small amount of Capilliary (endothelial cell nuclei) connective tissue -also note that is is also stained BROWN  Adipocytes/Adipose cells/Fat cells -they contain large lipid droplets that are unstained by either Hematoxylin or Eosin A. A. B. B. Figure 13. Epididymis. Has 2 (A) Golgi apparatus and (B) connective tissue in between Figure 11. Adipose tissue. Where (A) is the nucleus and adipocytes (B) contains large lipid droplets that cannot be unstained by H&E PREPARED AND EDITED BY: LIMBAUAN, J., LIVED, R., LOCQUIAO, C., LOPEZ, F., PADOLINA, J., PALAGANAS, B., PANG-AG, L.  (001) HISTOLOGY LAB DR. CABUENA | 09/14/2020  Mitochondria -rod-shaped, 0.1-0.2 µm in diameter (resolution limit of light microscope) -can be distinguished as darkly stained, thread-like structures in the apical cytoplasm of most cells. A. Figure 14. Small Intestines’ surface villi. (A) Mitochondria appear as dark grey lines that runs parallel to the long axis of the cell and perpendicular to the cell surface. TEST YOUR KNOWLEDGE 1. In a light microscope used for histology, resolution and magnification of cells are largely dependent on which component? A. Condenser B. Objective Lens C. Eyepieces or ocular lenses D. Specimen Slide E. The control for illumination intensity 2. Which of the following staining procedures relies on the cationic and anionic properties of the material to be stained? A. Enzyme histochemistry B. Periodic acid – Schiff reaction C. Hematoxylin & Eosin Staining D. Immunohistochemistry E. Metal Impregnation Techniques Answer: 1. B 2. C REFERENCES Mesher, A (2016). Junqueira’s Basic Histology Text and Atlas. McGraw-Hill Education. PREPARED AND EDITED BY: LIMBAUAN, J., LIVED, R., LOCQUIAO, C., LOPEZ, F., PADOLINA, J., PALAGANAS, B., PANG-AG, L.