M2 HISTO

• Stain used in the histology lab is hematoxylin & eosin (H&E)
• Steps and reagents used in the preparation of tissues for examination by a bright-field compound

  microscope:

  Fixation (formalin)
• Steps and reagents used in the preparation of tissues for examination by a bright-field compound

  microscope:

  Dehydration (ethanol)
• Steps and reagents used in the preparation of tissues for examination by a bright-field compound microscope:

  Clearing (xylene)
• Steps and reagents used in the preparation of tissues for examination by a bright-field compound microscope:

  Infiltration (paraffin)
• Steps and reagents used in preparation of tissues for examination by a bright-field compound

  microscope:

  Sectioning (rotary microtome)
• Steps and reagents used in preparation of tissues for examination by a bright-field compound

  microscope:

  Staining (hematoxylin and eosin)
• Steps and reagents used in preparation of tissues for examination by a bright-field compound

  microscope:

  Mounting (resin)
• Clearing agents:

  Acts as an intermediary between the dehydrating agent and the

  embedding agents, and are miscible with both.
• Clearing stage makes the tissues translucent
• Infiltration stage

  Stabilize the tissue using liquid paraffin in preparation to cutting.
• The fully cleared tissue is then placed in melted paraffin in an oven at 52 - 60 °C which

  evaporates the clearing solvent.
• The paraffin-embedded tissue is placed in a small mold with melted paraffin and allowed to

  harden at room temperature.
• Embedding prevents distortion of tissue structure during microtomy.
• Trimming

  Removal of excess wax to expose the tissue for sectioning on a microtome.
• the trimmed wax shape should be Truncated Pyramid
• Thin sections (5-15um) are cut

  using a microtome.
• A rotary microtome is

  usually used for

  paraffin sections.
• The paraffin block is taken from the mold and attached to a chuck that can be placed on the

  microtome.
• The block is trimmed to appropriate size, placed on the microtome, and a ribbon of sections

  (of 3-5um thickness) are cut.
• The ribbon is placed in a tissue-floatation bath carefully, to smooth out any wrinkles in the

  ribbon.
• The temperature of the bath should be 10 °C less than the melting point of the paraffin.
• Fixation is to preserve tissue structure
• The most commonly used fixative is 10 % neutral buffered form (NBF – phosphate buffer)

  formalin.
• fixation can prevent autolysis

  which is when enzymes inside the cells that could destroy the cells itself
• Fixation Hardens tissue by cross-linking proteins
• Formalin is a 37 – 40% (saturated) aqueous solution of formaldehyde gas.
• Formalin Acts upon the proteins that harden/stabilize the tissue.

  Doesn’t react with lipids and carbohydrates.
• Dehydration is when water has to be removed from the tissue.
• Direct use of 100% ethanol will cause cell damage.
• sections are picked up onto clean albuminized (adhesive) slides.

  when egg whites are used in the slide as an adhesive
• Universal size of standard slides is 76x25mm, with a thickness of 1.0-1.2mm.
• Overnight drying at 37 °C is used for many tissues.
• Most cells and extracellular materials are completely colorless, and to be studies

  microscopically all tissue sections must be stained.
• Nucleic Acid

  o Negatively charged

  o Has an affinity to basic dye
• basic dye is also termed as basophilic
• Cytoplasm Organelles Proteins

  o Positively charged

  o Has an affinity to acidic dye
• acidic dye is also termed as acidophilic
• Before staining the paraffin has to be dissolved out of the tissue using xylene or toluene.
• Hematoxylin Is soluble in water