Tissue Prep/Staining Flashcards
Fixing
- prevents deterioration, and hardens tissue.
- formalin is most common: reacts with amino acid to stabilize tissue structure, not good for detail
- all contain glacial acetic acid: counters shrinkage
Basic Fixing
- used for mitochondrial staining.
- chromatin is dissolved
Why must sample be dehydrated?
-will be embedded with paraffin (hydrophobic material)
How is a sample dehydrated?
- series of more concentrated ethanol baths
- will destroy neutral fats
What happens when a sample is cleared?
-alcohol is replaced with xylene or cedar oil
What does clearing do?
Removes the paraffin embedded medium.
List three clearing agents.
- xylene
- cedar oil
- carbon tetrachloride
What is embedding process?
- specimen moved through three melted paraffin baths.
- placed in mold, filled with melted paraffin; after final bath.
- placed in cold water bath for rapid hardening.
How would you prepare thin slice of tissue?
- sectioning: fixed rotary microtome makes slices in fixed distances
- sharp razor and tubular holder will produce similar results
Why must we stain tissue?
-generally tissues are colorless
Steps involved with staining.
- remove paraffin from the slide mounted section with xylene.
- remove xylene with graded [alcohol] down to water.
- apply stain.
- dehydrate with series of alcohol
- remove alcohol with xylene
- cement slide together.
Hematoxylin and eosin
- used to display structural features.
- not much about chemical characteristics of the tissue.
Eosin
- stains cytoplasmic components and extracellular
- yellow to pinkish
What do orcein and resorcin fuchsin stains reveal?
-elastic material
Silver Impregnation
-show reticular fibers and basement membranes
Lipids
- sudan will show the lipid.
- preservation requires technique without use of alcohol (dehydration)
How do basic dyes cause staining?
- react with anionic groups in tissue.
- phosphate, sulfate, carboxyl.
- higher pH allows more availability for binding.
List examples of basic dyes.
- methyl green
- methylene blue
- pyronine G
- toluidine blue
How do acid dyes bind?
- form electrostatic linkages with cationic groups. amino groups
- use in sequence to provide different results.
Acid dye examples.
- acid fuchsin
- aniline blue
- eosin
- orange g
Metachromasia
-dye changes color after reacting with tissue
What are histochemical staining techniques used for?
-study chemistry of cells and tissues
Schiff reagent reaction
-reacts with aldehyde groups to form a deep-pink color after exposure to HCl
Periodic acid-Schiff reaction
-periodic acid cleaves bonds of carbohydrates to form aldehyde group
How can RNA organelles be stained?
- use of basic dyes
- require control slides to note basophilic substances
Immunocytochemical techniques are used for?
-detect the presence of antigens by monoclonal antibodies
Antigens
- proteins
- glycoproteins
- proteoglycans
Antibodies
-activated B cells form monoclonal antibodies ( specific)
How are antibodies stained?
- fluorescent dye
- conjugated with visible substance for light microscopy
- gold or ferritin to produce visible marker in TEM
What is indirect labeling?
-marker attached to second antibody, specific to antibody which located the antigen of interest.
Steps taken in order to visualize tissue on a slide.
- fixing
- dehydration
- alcohol removal
- embedding
What is the process of staining?
1) remove paraffin with xylene.
2) remove xylene with gradient [alcohol]
3) apply stain and dehydrate with [alcohol]
4) clear alcohol with xylene
5) cement slide to prevent changes
What is a hybridoma?
-Fusion of activated B cell and myeloma.
Acid Fixative
-fix chromatin, nucleoli, spindle fibers
Carnoy’s Fluid
Acid fixative. alcohol, chloroform, glacial acetic acid. Preserves glycogen.
Zenker’s Fluid
Acidic. potassium dichromate, mercuric chloride, glacial acetic acid. Used for high detail.
Bouin’s Fluid
picric acid, formalin, glacial aceitc acid. Prolonged washing process.
Zirkle-Erliki Fixative
potassium dichromate, ammonium dichromate, copper sulfate, distilled water.
