Intro to Histological Techniques ✅ Flashcards
Histology
Study of fine detail of biological cells and tissues
Uses microscopy to look at specimens of tissues prepared using histological techniques
Histology basic def
Microscopic study of normal tissues
Histopathology
Study of pathology in tissue
Pathology
Study and diagnosis through examination of surgically removed -organs,
-tissues (biopsy samples)
-bodily fluids
-sometimes- whole body (autopsy)
Biopsy
Procedure
To remove a piece of tissue or sample of cells from body
-so it can be analyzed in a lab
Tissue prep name
Paraffinization
Paraffinization steps
- Fixed
- Washed
- Dehydrated
-in a series of alcohol solutions (increasing concentration) - Cleared
-xylol or toluol to remove alcohol - Embed in melted paraffin
- Sectioned
- Stained
*steps 3-4 done with histokinette- automated step
Fixation
37% formaldehyde solution
-most common fixative
-tissues structure not altered
-generates weak H-bonds between proteins
Embedding
Solidified paraffin block trimmed to appropriate size & sectioned with slicing machine (microtome)
- dispense wax
- align tissue
- cool in place
- ID bead
- top-up wax
6.cool plate
Frozen tissue prep
For short term & faster use-> can rapidly freeze specimens
-instead of paraffin embedding (OCT compound in liquid N)
Cryostat used for cutting sections
Integrity isn’t great if frozen too fast
-the membranes break open
What can you do with tissues that are to be sectioned frozen?
- Frozen UNTREATED- in liquid N
- Sectioned
- Stained- mostly H&E staining
-usually done for fast assay (in operating theatre) - Fixed w mild fixative
- Cryo-protected in sucrose (10%, 20%, 30% sucrose)
- Embedded in OCT
- Frozen in -80 C
- Sectioned
- Stained- used for IHC or IF (these methods use specific antibodies against protein under investigation)
*IHC= immunohistochemistry
**IF= immunofluorescence
How thick are the tissue sections in:
1. Optical microscope
- EM?
- 5-10 microm
- 50-100 nm
Histochemistry
Histo + chemistry = chemistry in tissues
-one of most widely used techniques to help scientists localize and visualize cellular components, tissues and other living structures
-using diff stains and indicators (which react w the cellular components) to develop tiny colored structures that could be easily observed under a microscope
Acidic dyes in Histochem
Neg net charge on colored portion
React with + (catatonic groups) cell and tissues- particularly w amino groups of proteins
ACIDOPHILIC
Basic dyes in Histochem
Pos net charge on colour end portion
React w anionic components (-) of cell and tissues
-phosphate groups on nuclei acids
-sulphate groups of gylcosaminoglycans
-carboxy terminal of proteins
BASOPHILIC
Common dyes
Haematoxylin & Eosin
-most common
-mostly used to display structural features of the tissue
Haematoxylin
Purple basic dye
Stains acidic macros
-DNA (nuclei),
-RNA (cytoplasm of cells with many ribosomes eg in glands),
-some proteins
Pink acidic dye
Stains basic macros
-muscle cell proteins,
-collagen (a major extracellular protein),
-cytoplasm of most cells
IHC
Visualization of distribution & localization of specific proteins in cells and in their proper histological context
Through indentification of antigens (proteins) using antibodies- that specifically bind to presented antigens
Types
1. Chromogenic IHC
2. Fluorescence IHC
Chromogenic IHC
Specific binding of primary antibody
Secondary antibody conjugated to an enzyme eg HRPO
Addition of substrate eg DAB will generate a colored precip
-used to indetify specific molecules in the tissue eg proteins, usually overexpressed in pathologies
Hybridization techniques
Specific sequences of RNA or DNA can be detected by hybridizing it with a fluorescently-labeled complimentary probe
Takes place in cell -in situ
Probes can be radioactively or fluorescently labeled
FISH- fluorescence in situ hybridization is used in genetic testing
Light microscopy types
A. Normal light
i. Bright field
ii. Phase-contrast
iii. Dark Field
B. Ultraviolet light
i. Fluorescence
C. Laser beam and scanning
i. Confocal
Electron microscopy types
A. Electron beam
i. Transmission Electron Microscope (TEM)
ii. Scanning Electron Microscope (SEM)
Phase contrast microscopy
Enhance the contrast of images of transparent and colourless specimens
Allows the examination of unstained cells and tissues
–very useful for the examination of living cells
