Intro To Histo Flashcards
Methods of tissue collection
- Needle biopsy (for almost any part of body)
- Endoscopic biopsy (any place accessible through openings in body)
- Transvascular biopsy (available for large blood vessels via catheter)
- Direct excision biopsy (surface of body via scalpel)
- Curettage biopsy (scraping of endometrial lining of uterus)
Tissue prep for light microscopy involves:
- Fixation
- Dehydration and clearing
- Embedding
- Sectioning
- Mounting and staining
Methods of fixation
Chemical: Formaldehyde, glutaraldehyde
- causes loss of cell activity
- cross links proteins to arrest changes
Dehydration by alcohol
Rapid freezing by liquid pentane at -160 degrees C
- halts all biological activity
- freeze until needed
What color does hematoxylin stain, and is it acidic or basic? What does it stick to? What is a component that likes hematoxylin called?
- Stains blue; is a BASE
- Acidic
- Basophilic
Types of stains (how they differentiate):
- Differentiate b/w acidic and basic components of cell
- Differentiate fibrous components of extracellular matrix
- Metallic salts that precipitate on tissues to form metal deposits on specific ones (good for TEM)
What color does eosin stain, and is it acidic or basic? What does it stick to? What is a component that likes eosin called?
- Stains pink; is an ACID
- Bases
- Acidophilic
What does toluidine blue stain? What color?
Stains polyanion-rich tissues PURPLE
What parts of the cell stain blue with hematoxylin?
Nucleus, RNA, DNA, parts of the cytoplasm rich in ribosomes - these are all ACIDIC structures and respond to base of hematoxylin
Characteristics of PARAFFIN WAX for embedding:
- What type of microscopy?
- Advantages?
- Disadvantages?
- How thin are the sections?
- Light microscopy
- Good resolution of cell structure and tissue architecture
- Slow (24 hours)
- 5-8 micrometers
Characteristics of ACRYLIC RESIN for embedding:
- What type of microscopy?
- Advantages?
- Disadvantages?
- How thin are the sections?
- High resolution light microscopy; Electron microscopy
- VERY thin sections w/ high resolution
- Doesn’t work with most histological stains; slow (several days)
- 1 micrometer for light, 60-80 nm for EM
Characteristics of FROZEN SECTIONS for embedding:
- What type of microscopy?
- Advantages?
- Disadvantages?
- How thin are the sections?
- All (?)
- Ideal for many stains; enzymatic activity reanimates once thawed out; rapid (minutes/hours)
- None
- THICK sections: 12-20 micrometers
Methylene blue and eosin together form:
Wright’s stain, used for differential staining of blood cells
What does Wright’s stain do, and what colors does it stain?
Differential staining of blood cells: RBC are pink, WBC are blue; both dyes cross cell membranes
Resolution of the human eye
0.1-0.2 mm
Resolution of light microscope
0.25 micrometers
Resolution of transmission electron microscope
0.2 nm
Resolution of scanning electron microscope
10 nm
List the types of microscope in order from worst (largest) to best (smallest) resolution.
Human eye < light microscope < scanning electron microscope < transmission electron microscope
What is a large disadvantage of histological sections?
They are not always representative: they are highly dependent on the method in which they are sliced. They are 2-D views of 3-D structures
What color does the nucleus of a cell stain?
Blue
How big is an erythrocyte?
7.5 micrometers
What can red blood cells be used for in the context of a histological sample?
Scale in a histological sample
What does PAS stain?
Carbohydrate-rich components of tissues and cells (uses H&E)
What type of visualization technique is autoradiography?
Allows us to see metabolic activity in tissues: inject radioactive precursor into individual, where it’s incorporated into macromolecules, which is sampled and viewed by overlay of film emulsion. Radioactive isotopes appear as silver granules.
What type of visualization technique is enzyme histochemistry? How does it work?
Can preserve enzyme activity in the tissue: provide enzyme w/ substrate that, when cleaved, deposits a colored product at the reaction site.
What type of visualization technique is immunocytochemistry? How does it work?
Identifies location of specific macromolecules by binding labelled antibodies to antigens on them.
What are the 2 types of immunocytochemistry?
Direct - antibody itself is labeled, recognizes antigen
Indirect - Send in primary antibody to bind to antigen, then send in labeled secondary antibody to bind to primary
What are the advantages of indirect immunocytochemistry?
Good for amplification b/c multiple binding sites on primary antibody AND secondary antibody can bind to several kinds of primary
What is the mechanism of transmission EM?
Using short wavelength electrons to visualize
What are two stains that would work for TEM?
Heavy metal salts like URANYL ACETATE and LEAD CITRATE
What is SEM used to look at, if not sections through a material?
Surface structures by 3D images
How do you prepare an SEM specimen?
Coat surface of specimen w/ heavy metal, then add enzymes that etch away real specimen, leaving a replica etched into atomic gold and/or palladium.
How does SEM work?
Etching is scanned through an electron microcscope, which analyzes electrons reflected off surface of heavy metal replica.
