Microscopy, Simple Special Stains Tutorial PDF

Microscopy, Histological stains Tutorial 2025  Learning Objectives identify different types of staining techniques. Explain the principle of staining with hematoxylin and eosin (H & E). Enumerate some special stains & their uses. Define histochemical , immunohistochemical stains...

Microscopy, Histological stains Tutorial 2025  Learning Objectives identify different types of staining techniques. Explain the principle of staining with hematoxylin and eosin (H & E). Enumerate some special stains & their uses. Define histochemical , immunohistochemical stains and their applications. compare between LM / EM sample processing.  Do you remember the steps of staining paraffin section??? I can not see  Paraffin technique (routine histological section) --------------------------------- 1. Selection and obtaining tissue. 9.Deparaffinization 2. Fixation. 10. Hydration. 3. Washing. 11.Staining 4. Dehydration. 12. Dehydration. 5. Clearing. 13. Clearing. 6. Infiltration and Embedding. 14. Mounting. 7. Microtomy and Sectioning. 15. Cleaning & 8. Mounting. Labeling.  staining technique 1- Deparaffinization Parraffin is removed by xylene for easy staining. 2- Hydration Passing the slide in descending grades of alcohol(100% - 90%- 70%-50%)then distilled water Why? Hydration of the tissue is a necessity to enable the stain reach and react with the tissue components  staining technique 3-Staining The slide is placed in Haematoxylin stain then washed with water then placed in eosin stain and washed with water. 4- Dehydration : by ascending grades of alcohol 5- Clearing: by xylene 6- Mounting: tissue is covered by cover slip. 7- Clearing & labeling Manual Slide Staining Programmable Automatic Slide Stainer  Some histological stains used for light microscopy  Basic stain vs acidic stain vs neutral stain ……… According to the chemical component of the stain, stains can be divided into: - Acidic stain - Basic stain - Neutral stain  Basic stain - Carries positive charge e.g: Hematoxylin, Methylene blue - stains acidic structures (as DNA-RNA-rER-nucleus) - Gives blue color (basoplilia) Acidic stain - Carries negative charge. eg: Eosin - Combines with basic structures (as proteins ,membranes, cytoplasm, collagen) - Gives pink color (acidophilia or esinoplilia) Hetamoxylin-eosin (H&E): In histological technique is probably the most used staining procedure for paraffin sections Basoplilic nuclei stained with hematoxylin Acidophilic cytoplasm stained with eosin  Neutral stain - When an acidic dye is combined with a basic dye a neutral dye is formed. - It stains all components of tissue but with different colors. - It used to stain the blood elements in a blood film. Examples: Leishman’s stain  Vital stain vs supravital stain……… What are the Similarities Between Vital and Supravital Staining? Vital and supravital staining are two staining techniques to examine living cells.  Vital stain It is the staining of the living cells within the living animal (i.e. in vivo). It is done by injecting a nontoxic dye into living animals. The principal|: is that the particles of stain are engulfed by macrophages in the cells (The uptake of dye is due to phagocytosis). e.g. staining of phagocytic cells with trypan blue or Indian ink.  Supravital stain It is the staining of living cells outside the body (i.e. in vitro). E.g: (a) The most (a) common supravital stain is performed on reticulocytes using methylene blue or brilliant cresyl blue. (b) staining of mitochondria with Janus green B. (b)  Trichrome stain Three stains are used in combinations to give 3 colors to different tissue components. In addition to staining the nuclei and cytoplasm can differentiate collagen from muscle as Masson’s stain: it stains collagen green nuclei blue Muscle red  Metachromatic stain -It is the staining of certain cell components with a color which is different from that of the dye used. -The phenomenon is called (metachromasia). -It is due to the interaction between the basic dye and the cell component, producing a different compound with a different color. -Exp: the staining of mucopolysaccharide granules of mast cells toluidine blue which changes to the violet color.  Histological stains used for electron microscopy  A- Transmission electron microscopy specimen - The most widely used stains in ultrathin sections of transmission electron microscopy are the heavy metals. - The double contrast method of ultrathin sections with uranyl acetate (UA) and lead citrate is the standard contrasting technique for electron microscopy - All the original electron microscopy images are in gray scale, from white to black. N.B: in EM: we use double fixation to preserve ultrastructural details (glutraldhyde then, osmium tetraoxide) Osmium preserve the tissue and stains lipids.  TEM photomicrograph  Histochemistry & Cytochemistry Principle: methods for localizing substances in tissue depending on chemical reaction between cell components and stain. The end product of the reaction is permenant, colored precipitate that can visualized under microscope. Examples: Demonstration of nucleic acids, enzymes, glycogen, Lipids. Exp: Enzyme Histochemistry Detection of ATPase enzyme ATPase detection in myosin fibers of skeletal muscles Exp: Carbohydrate Histochemistry - Detection of Tissue Glycogen by periodic acid schiff reaction: (PAS stain) Carbohydrate detection by PAS reaction gives magenta color Exp: Carbohydrate Histochemistry - Detection of Tissue Glycogen by Best’s carmine stain glycogen stains red Exp: Lipid Histochemistry - Detection of Tissue lipids by fresh or frozen section & staining with a- Sudan III (orange color ) b- Sudan black (black color) c- Osmic acid (black color) Sudan III Sudan BLACK Exp: Nucleic acids Histochemistry Detection of Nucleic acids (DNA) by feulgen reaction: DNA: red to purple  Immunohistochemistry Immunohistochemistry is a research method combining morphology and biochemistry to identify specific antigen. The principle: reaction between "antibody" that specifically binds to an "antigen", and "visualize" the site of reaction microscopically in the tissue. Microscopy  TYPES OF MICROSCOPES A) Depend on ordinary visible light 1. Conventional light microscope. 2. Polarizing microscope. 3. Phase contrast microscope. 4. Dark-field microscope. B) Depend on other than visible light 1. Fluorescent microscope. 2. X-ray microscope. 3. Laser microscopes. 4. Electron microscope. Conventional light microscope  Electron microscope Transmission Electron Microscope Scanning Electron Microscope Transmission Electron Microscope Scanning Electron Microscope  Comparison between light and electron microscope LM EM Source of illumination Daylight or electric Beam of electron light Condensers and Glasses Electromagnetic lenses lenses lenses Resolution 0.2 micron 0.2 nm power Magnification Up to 4,000 Up to 400,000 Images Colored image Not colored which of the following would be best suitable to visualize lipid? a. Masson's trichrome stain b. Silver impregnation c. Sudan stain d. Hematoxylin and eosin stain ------------------------------------------------------------------------------------------------- --------------------------- Hematoxylin: a. is a basic dye b. is a immunohistochemical stain c. stains basic components of cytoplasmic proteins d. stains collagen blue e. stains nuclei red Vital stain is a- staining of dead tissue outside the body b- staining of a living cell inside the body c- staining of a living cell outside the body d- staining of a dead tissue inside the body ------------------------------------------------------------------------------------------------------- --------------------- Fixation of fresh tissue is essential to a- remove water b- prevent putrefaction and autolysis c- replace alcohol d- clear the sections e- keep the cells alive