Slide Preparation and Microscopy Flashcards
Fixation
preservation of the specimen in tissue preparation. This is done with formalin, which is 37% formaldehyde
Embedding
sectioning in tissue preparation to allow for thin slides.
Mounting
attatching specimen to slide for better viewing. This is done using heat or an adhesive
Staining
distinguishes between tissues for slides
Steps of slide preparation
- Fixation
- Embedding
- Mounting
- Staining
Properties of formalin
37% formaldehyde. Formalin allows proteins to still react
Steps for Embedding
- Dehydration
- Clearing of alcohol
- Paraffin application
What is important to know happens during embedding?
Lipids are lost. They will show as blank spaces in the slide to show where they were
Commonly used dyes for staining
H&E and PAS
What is H & E staining?
Staining done using Hematoxylin (an acid) or Eosin (a base)
What is PAS?
Periodic acid-Schiff reaction
What elements are stained by acids?
Proteins and extracellular fibers like collagen
Eosin
an acidic dye that shows up as red in slides
Acidic dyes attract _____
basic structures with a positive charge
What is hematoxylin
a classic basic dye that shows up blue/purple in slides
Basic dyes attract _____
acid structures with a negative charge
What elements are stained by bases?
Heterochromatin, DNA, RNA, endoplasmic reticulum
What is the Feulgen reaction?
stains DNA. It distinguishes between DNA which reacts and RNA which does not react
The PAS stains what structures what color?
basement membrane and glycogen, which become pink
What color are elements stained with a basic dye?
blue/purple
What color are elements stained with an acidic dye?
Red/pink
What cellular structures does Alcian blue stain?
Mucin which becomes light blue
What cellular structures does Azan stain?
Nuclei, red blood cells, and muscle; bright red.
Also stains mucin and basement membrane; light blue
What cellular structures does Congo red stain?
Amyloid which becomes red
What cellular structures does Giemsa stain?
red blood cells which become pale pink, and nuclei with become blue
What stain is used to differentiate between human and bacterial cells?
Wright stain
What stain is used to differentiate between muscle and connective tissue?
Masson’s Trichrome
3 colors of Masson’s Trichrome + what each color stains
Blue; nuclei
Blue/green; connective tissue
Red; keratin, muscle, and hyaline
What does orange G stain?
Red blood cells which are stained orange
What does Sudan black & osmium stain?
Myelin which is stained brown/black
What stains mucus/mucopolysaccharides in the cornea?
Alcian blue
What stains amyloid in the cornea?
Congo red
What can be diagnosed with alcian blue staning in the cornea?
Macular dystrophy
What can be diagnosed with congo red staining in the cornea?
Lattice dystrophy
What stains hyaline in the cornea?
Masson’s trichrome (it becomes dark red)
What can be diagnosed with Masson’s trichrome staining in the cornea?
Granular dystrophy
What type of dystrophy is a combination of lattice and granular dystrophy?
Avellino dystrophy
What two dystrophies are Avellino dystrophy a combination of?
Lattice and Granular dystrophy
What stains carbohydrates in the cornea?
PAS (periodic acid-Schiff) (becomes pink)
What can be diagnosed with PAS staining in the cornea?
Fuch’s endothelial dystrophy
Enzyme digestion
Results in structures disappearing after digestion
Enzyme histochemistry
reagent causes a colored precipitation product
Innumocytochemistry
antibodies bind to components of the cell. This can be detected by fluorescein tagging
Direct antibody binding
the antibody that has been tagged to fluorescein will attach directly on to the antigen
Indirect antibody binding
the antibody is not tagged but will bind to the antigen. Then a tagged antibody will bind to the untagged antibody (could result in multiple antibodies tagged to each other)
How is hybridization detected?
Via a radioactive label or fluorescein
What is autoradiography?
Labelling of precursors with a radioactive label
micrometer
μm (10^-6)
nanometer
nm (10^-9)
millimeter
mm (10^-3)
picometer
pm (10^-12)
How does atomic force microscopy work?
Uses a physical problem on live cells to scan the specimen
Methods of Embedding
Use frozen sections or paraffin embedded specimens
Tools for embedding
The tissue is sectioning using a microtone or a steel knife
