Intro to Histology Flashcards
Light microscopy
- pass visible light through a specimen
- lens allow both magnification & resolution of details within the tissue specimen
what is resolution?
-ability to distinguish two close but distinct points
Steps to prepare tissue for imaging (6)
1) fixation
2) dehydration
3) embedding
4) sectioning
5) removal of resin/parafin
6) staining
what is fixation?
- stabilizes structures & prevents degradation
- use alcohol, paraformaldehyde
what is dehydration?
the gradual removal of H20 in preparation of tissues
- allows the auditioning of embedding w/ a hardening agent
- use a series of baths with progressively increasing alcohol proportions
what is embedding?
- infiltrate the tissue w/ harder substance to make it easy to cut into thin sections (resin/parafin)
- keeps tissue from shriveling up
What is sectioning?
-tissue is sectioned using rotary microtome so that the tissue slice is translucent and can be seen through w/ the microscope
what is straining?
-the introduction of contrasts to tissue that is otherwise transparent
what is cryo-alternative?
- used for immediate evaluation of tissues & immune-fluorescence techniques
- fast/easy alternative since doesn’t require dehydration or embedding
- uses deep freezing
- but less stable than parafin/resin embedded sections
benefits of Cry-alternative process? negatives?
1) rapid freezing reduces ice crystal formation & preserves cell structure
2) tissue not as stable/ doesn’t last as long
- exspensive
What are cell smears?
- blood or bone marrow epithelial cells which are not sectioned, spread from source directly onto the glass sheet
- are often fixed in alcohol (either bath or sprayed)
- are processed for staining immediately
What is Hematoxylin and Eosin (H&E)?
hematoxylin: a basic dye that stains acidic components of cells blue (nuclei, RER, DNA/ribosomes)
eosin: an acidic dye that stains the basic components of cells a reddish-pink (cytoplasm, bone matri, proteins)
How H& E used to ID midoticallly active cells?
- euchromatin is DNA in use, it is spread out/diffused and less stained since open
- heterochromatin (compact) is condensed/ replicating cells and stains darker
Periodic acid-Schiff (PAS) staining
- used for carbohydrates, polysaccharides (glycogen) and gut/liver sections
- pink/magenta color gives very clear lines
H&E vs H& PAS?
- H& PAS gives much clearer/distinct lines since are lighting up polysaccharides
- mixed with hematoxylin to label nuclei blue
orecin staining?
used to stain elastic fibers dark brown/purple
-example is the elastic component in the walls of arteries or in the matrix of elastic cartilage
Osmium tetroxide?
-stains lipids & myelin dark black
Oil red O?
- stains neutral lipids a red-orange color in unfixed frozen sections
- since fixation dissolves lipids
Toluidine blue staining
- high affinity for acid tissues-stains nucleic acids blue & polysaccharides purple
- good for chromosomes
metal impregnation?
- silver is most commonly used to demonstrate reticular fibers/neurons
- tissue bathed in metal solutions and metal will attach to specific cell components
nissl stain?
- selective staining, uses an aniline stain to label extra-nuclear RNA granules
- nucleic acid staining method use don nervous tissue sections
- binds (-) charge on nucleic acid
- marks ER as well
what is vital staining? (4 types)
- the uptake of dyes by the cell
1) trypan blue
2) Wright (Giemsa stain)
3) Neutral Red
4) Janus Green
Trypan blue?
- rapidly engulfed by macrophages and can be used to delineate liver cells
- DISTINGUISHES alive/dead cells
Giemsa staining (Wright)
mixture of methylene blue, eosin and azure Blue
- attaches to DNA
- stains blood/bone marrow smears & parasites
neutral red
- incorporated by live cells into their lysosomes
- red when protonated
- colorless and permeable when unprotonated
- accumulate in acidic lysosomes where protonation is occurring
Janus green?
- changes color w/ different amounts of oxygen
- green= oxidized
- colorless= before oxidation
- accumulates in oxidative env. of mitochondria
What is immunohistochemistry (IHC) or immunocytochemistry (ICC)? Process?
- use of antibodies to label proteins of interest
1) antibodies either directly labeled or need secondary antibody
2) use fluorescent dyes/enzymes that enable a colorimetric rxt
IHC vs ICC difference?
-IHC= uses tissue sections ICC= staining of isolated blood cells or cultured intact cells
Two types of electron microscopy?
1) Scanning electron microscope (SEM)
2) Transmission electron microscope (TEM)
What is transmission electron microscope?
(TEM)
- form of electron microscopy
- electrons passed THROUGH specimen
- great for INTERNAL detail
- focused on magnetic lenses
- image formed on fluroescent screen then photoed
- better resolution than SEM
What is scanning electron microscope?
(SEM)
- electrons passed round the specimen
- provides detailed study of the SURFACE
- lower resolution than TEM
