PS1_Histo_Lect4_Part 2_Power Point Presentation.pptx

STAINING – MAIN TYPES OF ACTION Tinctorial Give tissues the colour of the reagent. Histochemical colourless product. A chemical reaction on a slide, often solutions produce a coloured end Enzyme histochemical Enzymes in the tissue react with added substrate and a chromogen to produce a coloured in...

STAINING – MAIN TYPES OF ACTION Tinctorial Give tissues the colour of the reagent. Histochemical colourless product. A chemical reaction on a slide, often solutions produce a coloured end Enzyme histochemical Enzymes in the tissue react with added substrate and a chromogen to produce a coloured insoluble end product. Immunohistochemical Antibodies bind selectively to antigens in cells and/or tissues. Visualised with a chromogen (DAB) or A bound fluorochrome (FITC) – bright fluorescence BASED ON CHEMICAL NATURE: ACIDIC, BASIC AND NEUTRAL STAINS. Acidic Dye/Stains  Owe their colour to the anion (negative charge), with sodium usually being the cation.  Examples: Eosin, Carbol Fuchsin, India ink Basic Dye/Stains  Owe their colour to the cation (positive charge), with the anion (negative charge) commonly being a chloride.  Examples: Chrystal violet, methylene blue, safranine Neutral (compound) Stains  These dyes are often referred as the blood dyes, in recognition of composition of Romanowsky stain (eosin/methylene blue). Acid and Basic elements are combined also to stain ‘neutral’ elements.  Examples: Giemsa, Leishman and Wright’s stains. Haematoxylin is an acid dye but is combined with a mordant to create a ‘dye lake’ which is basic (dye radical on +ve charge) MECHANISM OF STAINING STAINING – OVERALL AIMS Introduction of contrast into cellular preparations Highlight areas of interest = specific targets Aim for: high degree of selectivity high degree of sensitivity Minimal background staining WHY IS STAIN RETAINED IN TISSUE?  After removal from the staining bath, stain retention occurs if stains have a high affinity for tissue elements and/or low affinity for processing fluids and mounting media, or if stains dissolve in these latter solvents slowly.  Sections stained with routine basic dyes must be dehydrated by either passing rapidly through the alcohols, using non-alcoholic solvents or by air-drying.  Dehydration is less critical with acid dyes. Sections stained with acid or basic dyes are usually mounted in non-aqueous media which do not extract dye.  Alternatively, dyes may be immobilized, e.g. by formation of iodine complexes in the Gram stain. Example of a section that was extensively dehydrated in alcoholic solvent and Eosin (pink) WHY ARE STAINS NOT TAKEN UP INTO EVERY PART OF THE TISSUE?  Numbers and affinities of binding sites  Rate of reagent uptake  Rate of reaction  Rate of reagent loss  Metachromatic staining HOW IS STAINING INFLUENCED BY TISSUE FIXATION?  Fixation is carried out to reduce the non-vital tissue autolysis causing morphological changes, and to prevent losses of some tissue constituents in the processing and staining solutions.  A given substance may be retained in the specimen to different extents by different fixative agents, and nothing can be stained if it is not retained.  For example, many lipids are well preserved after fixation in osmium tetroxide or dichromates, but are poorly preserved after formalin fixation. However, lipids are actively extracted during alcoholic or acetone fixation. Thus, staining lipids after alcoholic fixation is ineffective.  For example, alcohol and acetone, although poor at retaining proteins, are also poor at destroying the activity of whichever antigen or enzyme is retained. Both retention and reactivity of substances affect staining, and both may be fixative dependent. WHAT ARE THE EFFECTS OF SPECIMEN GEOMETRY STAINING? ** Here theON ‘specimen’ refers to the biological material in contact with the staining solution, e.g. a dewaxed section • Typically, thin specimens achieve staining equilibrium faster than thick. • Specimens with irregular surfaces are stained faster than smooth surfaces, and dispersed cells faster than uniform slabs. • Complex geometries can arise when preparing smears from epithelia. These often contain multicellular clumps of cells, as well as monocellular dispersions. Cells in the middle of such clumps are less accessible to stains than the peripheral cells. • Geometrical complexity also arises from swelling of cell and tissue components in staining solvents. A modification of section geometry induced by microtomy is “chatter”. This artifact, related to poor section cutting, produces sections comprised of alternating thick and thin strips. Staining may then generate alternate strips of strong and weak colour DEMONSTRATION METHODS SENSITIVITY DEPENDS ON Retention of target in tissues = fixation dependent Preservation in a reactive form = fixation dependent Exposure during staining method = pre-treatment Selectivity of stain binding to target = pH, ions, mordant Enhancement of bound stain e.g. Ag techniques DEMONSTRATION METHODS ARE: 1. Designed to add contrast to sections 2. Designed to react with specific targets For example the Haematoxylin & Eosin (H&E) stain Targets the nucleus – haematoxylin Targets the cytoplasm – eosin DYE NOMENCLATURE STANDARD OPERATIONAL PROCEDURE (SOP) – FOLLOW THE INSTRUCTIONS  Staining protocols may wary between laboratories  Adjusted/changed  Training will be given  Competencies will be signed  John D Bancroft – all laboratories follow this book How would you prepare a saturated Congo Red Solution?  I will follow the SOP.