Histology Techniques: Flashcards
Describe the process of fixation in histology:
Preserve tissue structure by halting autolysis (destruction by own enzymes), decomposition and putrefaction (destruction by bacteria/ fungi)
Common types of fixative:
- 4% formaldehyde in solution used for routine examination
- glutaraldehyde for electron microscopy
- alcohol used for cytology
- acetone used for frozen histology and cytology
Over-fixation or under-fixation can distort tissue structure
Describe the fine-tuning of fixation:
Buffering - best at neutral pH, hypoxia lowers pH, buffers are used to prevent excessive acidity e.g. phosphate, bicarbonate, cacodylate,etc
Volume- should be a ratio of >10:1 fixative to tissue
Temperature- heating fixatives increases the speed of fixation
Concentration - too high a concentration may damage the tissue. E.g.
Formalin works best at 10% (ie 4% formaldehyde solution)
Time – as soon as possible, when not possible should be kept moist in a saline solution (PBS) and consider a quick fixative
Describe the process of embedding including errors/ artefacts produced:
Purpose - stabilise tissue for sectioning by encasing it in a supportive medium (e.g., paraffin wax or resin).
Process - dehydration (alcohol), clearing (xylene), and infiltration with embedding medium.
Ideal embedding membrane should be:
- soluble in processing fluids
- molten between 30 and 60 degrees
- translucent, colourless
- stable and homogenous
Errors:
- incomplete dehydration or clearing leads to poor embedding and damaged section
- overheating cooks tissues
- air bubbles can affect tissue
- improper orientation
Describe the process of sectioning:
Microtome - blade with a mechanical mechanism for advancing the block by small amounts between each section
Why do we stain structures ?
Most tissues and cells are colourless under the microscope
Used to highlight different structures
Experiments can be performed to highlight specific antigens
Stains have an affinity for cellular components
Describe the process of staining preparation:
Must remove wax with xylene
As most stains are aqueous, sections are then rehydrated by running them through a graded series of decreasing concentrations of alcohol to water
Describe the process of mounting including errors/ artefacts produced:
Purpose - preserve stained sections for microscopic analysis using a mounting medium (e.g., DPX) and a coverslip.
Errors:
- air bubbles
- excess medium can obscure tissue
- uneven mounting produces some tissue in focus and some not
Explain stains haematoxylin and eosin:
Haematoxylin:
- requires a metal cation (Fe, Al, W) ‘mordant’ to help it link to the tissues
- stains nuclei blue to purple
- binds to acidic components like nucleic acids
Eosin:
- stains cytoplasm, connective tissue and extracellular components pink
- binds to basic components like proteins
Describe the staining artifacts:
Uneven staining – improper
washing, uneven fixation or
inconsistent staining protocol
Over-staining – ‘toast effect’
Crystalline Precipitates –crystal formations from excess stain
Describe the microtome artefacts:
Knife Marks – lines or grooves in the tissue from dull/damaged blade
Chatter Marks – Ripple like artefacts caused by vibration during sectioning
Compression – Remaining wrinkles post sectioning (not long enough or not able
to overcome in water bath)
What are the fixation artefacts produced ?
Shrinkage– affects the size and shape of cells, changing morphology due to dehydration
Formalin Pigment– Black or brown pigment formed reaction with haemoglobin in poorly fixed tissue
Autolysis – degradation of tissue due to delayed or under fixation
Overfixation - too long in fixative makes it overhardened, makes sectioning challenging and distorts morphology
What are some advanced applications of histology?
Immunohistochemistry (IHC):
Detect specific proteins using labeled antibodies (e.g., HER2 in breast cancer).
In Situ Hybridization (ISH):
Localize specific DNA/RNA sequences (e.g., FISH for chromosomal abnormalities).
Electron Microscopy:
Ultra-structural analysis of cellular organelles (e.g., mitochondria).
