Tissue Prep & Staining Objectives Flashcards
What are the components of a light microscope and the pros cons?
Light source, condenser,stage,objective lenses and ocular lenses.
Pros: ability to magnify and resolve structural details
Cons: specimen must be thin and there is little contrast if not stained
How does a phase contrast microscope work and the advantages?
It converts phase shifts into visible changes in light. Useful for observing unstained cells or living cells.
How does a confocal scanning microscope work and the advantages?
Adds a pinhole at the confocal plane to increase optical resolution and contrast which eliminates any out of focus light.
Allows for the 3D construction of images as images are projected onto a screen for a visual image.
Combination of love gut and fluorescent microscopy- uses a laser beam.
Advantages: thin optical images are made, any out of focus image is deleted by computer, computer can stack images to form 3D image.
How does TEM work & pros and cons?
Uses a beam of electrons instead of light. Uses the potential difference btw a cathode and anode to affect voltage to drive electrons through column.
Cons: very expensive
Pros: great detail
Compare scanning electron with transmission electron.
Come back to.
What are the steps for fixing and embedding?
Fixing
Dehydration
Removal of alcohol
Embedding
What is fixing?
Fixing helps to harden the tissue and prevent further deterioration. Usually distorts the specimen.
-Formalin is most common, but not useful if
great detail is needed
Differentiate between acid and basic fixatives?
Acid can fix chromatin nucleoli ams spindle fibers.
- Carnoys fluid, Zenkers fluid,Bouins fluid
Basic can fix mitochondria, chromatic is dissolved.
- Zirkle-Erliki fixative - glutaraldehyde:cross links proteins
Describe Dehydration.
Water must be removed bc the tissue will be embedded with hydrophobic material (paraffin).
Place the tissue in increasing baths of ethanol
-ethanol dissolves neutral fats so N-
butyl alcohol or acetone can also be used
Clearing (removal of alcohol):
Replace the alcohol with xylene or cedar oil, as paraffin doesn’t mix with alcohol but will with these.
Embedding:
Move tissue through 3 melted paraffin baths to remove xylene. After 3rd bath the tissue is placed into a mold and hardened carefully by cold water bath.
What are advantages of rotary microtones over hand held ones?
Come back to.
How does tisssue sectioning from TEM differ from paraffin embedded specimens?
Diamond knives must be used and sections are 50 to 100 micrometers. Sections are placed on plastic grid coated in copper as they are too fragile. Electron beam passes through the holes.
In paraffin tissues sectioning a rotary microtome is used and a razor blade is used. The tissue is placed on a glass slide and a slip is placed over it for viewing.
What steps are needed for staining the paraffin section?
Paraffin is removed with xylene, and then xylene is removed with decreasing concentrations of alcohol to water. Next stains are added and then the section is dehydrated through alcohol again, and alcohol is removed with xylene. Finally a drop of cement is placed on section with a cover slip.
Compare basic and acidic dyes.
Basic dyes: reacts with anions (phosphate, sulfate, and carbonyl groups). Binding is dependent upon pH. Hematoxylin not basic but behaves like a basic dye. Methyl green, methylene blue, pyronine g, toluidine blue are basic. Anything that reacts with these is basophilic.
Acidic dyes: form electrostatic linkages with cationic groups such as AA of proteins. Acid fuchsia, aniline blue, eosin, orange G. Anything that reacts with these are acidophilic.
