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Lecture 43 Histology (Techniques), Tissue Preparation Steps, CONTRAST…
Lecture 43
Histology (Techniques)
Tissue Preparation
1. FIXATION
preserve structures
Penetration
: tissue cut into small sections
Most common:
37% Formaldehyde
Leads to
Insoluble macromolecular complexes
&
Dissolution
of soluble smaller molecules
2. PROCESSING
(2A)
Embedding
- paraffin/plastic resins
(2B)
Sectioning
- microtome/cryostat
(2C)
Mounting
- tissues laid gently on slide
3. STAINING
H&E
Hematoxylin= blue
Eosin= red
Silver Stain
for connective tissue
Nuclei=
dark blue!
Cytoplasm=
pink!
Periodic Acid-Shiff Reaction (PAS)
Schiff reagent (fuchsin)
Stains polysaccharides "PASs the SUGAR!"
amylase/diastase
Fuelgen Stain
Stains DNA; DNase abolishes
Cytochemistry
uses enzymatic activity to localize structures
(i.e., alkaline phosphatase, peroxidase)
Fe
detection
Immunohistochemistry
Antibody/antigen rxns
Monoclonal/polyclonal
Ab-conjugated to dye
fluorescin
Direct/indirect
Hybridization
In situ
; oligonucleotide probes
FISH
- uses fluorescent DNA/RNA probe bind to
specific
gene of interest on chromosomes
Autoradiography
radioactive materials exposed to tissue; tags certain things
4. MICROSCOPY
General Microscopy
Visible Light M
Bright field
tissue exposed to light ("
like X-ray
)
Dark field
NO staining needed
Scattered light collected --> greater detail!
Phase contrast
NO staining needed!
Denser areas appear more
Confocal
Surface Metrology
quantification of shape w/reflective light
surface topography
NO detection of
out-of-focus
light
3D images!
Fluorescence
Stain/label w/fluorescence using
fluorescein
Electron Microscopy
e- beam (1000x; 3nm)
10^6x magnification
Specimen:
-thin, small sections
-heavy metal stains
-excellent quality A+
dust-free environment
Transmission EM
Scanning EM
5. Interpretation
Artifacts
-plane of section hard to control
-->
limitations!
(preparation) shrinkage, cracks, empty spaces, etc.
3D tissue --> 2D sections
Tissue Preparation Steps
CONTRAST STAINING