Ex 1 TIssue Prep and Staining Flashcards
1
Q
steps of tissue prep
A
- fixing
- dehydration
- removal of alcohol
- embedding
2
Q
steps of tissue prep
A
- fixing
- dehydration
- removal of alcohol
- embedding
3
Q
Purpose of fixing a tissue specimen
A
prevents further deterioration of tissue specimen and helps to harden the tissue prior to embedding and sectioning
4
Q
What are the characteristics of the ideal fixative?
A
give greater optical contrast (with staining) with the least amount of distortion
5
Q
What is one of the most widely used fixing agents?
What are characteristics of it?
A
Formalin
- used alone or in combo with alcohol (shrinks tissues) and/or acetic acid (softens and counteracts shrinkage of alcohol)
- reacts with amino acids of the tissue proteins and stabilizes tissue structure to prevent further deterioration
- not good if fine cytological detail is desired
6
Q
What do acid fixatives fix?
A
chromatin, nucleoli, spindle fibers
do not fix mitochondria or nucleoplasm
7
Q
Carnoy’s fluid characteristics
A
- acid fixative
- mixture of alcohol, chloroform, and glacial acetic acid
- good general fixative
- used for preserving glycogen in animal tissues
8
Q
Zenker’s fluid characteristics
A
- acid fixative
- contains potassium dichromate, mercuric chloride, and glacial acetic acid
- used when sharp histological detail is desired but must be washed out to prevent precipitation of black crystals
9
Q
Bouin’s fluid characteristics
A
- acid fixative
- picric acid, formalin, and glacial acetic acid
- widely used gives good cytological detail
- requires prolonged and careful washing cycles
10
Q
Basic fixatives
A
- fix tissues where mitochondrial staining is desired
- chromatin is dissolved
11
Q
Zirkle-Erliki fixative characteristics
A
- basic fixative
- potassium dichromate, ammonium dichromate, copper sulfate, distilled water
- long fixing time (2 days) and washing under running water
12
Q
List of acid fixatives
A
Carnoy’s fluid
Zenker’s fluid
Bouin’s fluid
13
Q
Fixatives for TEM
A
- glutaraldehyde: preserves proteins by cross-linking them
- osmium tetroxide: reacts with lipids (especially phospholipids) and imparts electron density to cell and tissue structures
14
Q
Purpose of dehydration
A
Remove all water from tissue because it will be embedded and infiltrated with a hydrophobic material
15
Q
How to complete dehydration
A
place tissue in successively increasing strengths of ethanol
16
Q
Limitations of using ethanol, n-butyl alcohol, or acetone for dehydration?
A
dissolve neutral fats
17
Q
How is alcohol removed after dehydration and what is the purpose of removing the alcohol?
A
Replace with xylene or cedar oil so that paraffin (for embedding) can mix with it.
Tissue usually becomes transparent.
18
Q
Steps of embedding
A
- several sequential melted paraffin baths
- placed in mold and filled with with melted parafifin
- mold is hardened by placing in cold water bath
usually centimeter in diameter
19
Q
Embedding for TEM
A
- tissues are infiltrated with a monomeric resin (epoxy resin)
- resin is then polymerized
tissue samples are typically less than 1mm cubed
20
Q
How do you prepare a thin slice of tissue after it has been fixed and embedded?
A
Sectioning using a rotary microtome (usually)
21
Q
Sectioning
A
- smaller than 10um
- paraffin is removed when viewing
- rotary microtome or sharp razor with tubular holder
22
Q
Sectioning for TEM
A
- 50-150nm
- diamond knives
- sections are too fragile, floated onto a plastic-coated copper mesh grid (holes allow electrons to pass through)
- plastic left in place during view
23
Q
Why must tissues be stained following sectioning?
A
Animal tissues are usually colorless so they must be artificially colored
24
Q
How do you prepare tissue for staining?
A
- remove paraffin from section using xylene (section is mounted on slide)
- remove xylene using graded series of alcohol down to water
- apply stains and again dehydrate using graded series of alcohol
- remove alcohol with xylene
- add drop of cement (mounting fluid) and cover slip
25
Examples of stains
- H&E (hematoxylin and eosin)
- orcein and resorcin fuchsin stains
- silver
- Sudans
- basic dyes
- acid dyes
26
H&E stains
- commonly used for routine staining b/c they display structural features
- do not say much about chemical characteristics
- behaves like basic dye due to mordant that helps it bind to tissues
- hematoxylin is derived from logwood as hematein and stains nuclear material and some cytoplasmic (RER) blue or purple
- eosin is an acid dye, stains cytoplasmic components and extracellular material as yellow or pink
27
Orcein and resorcin
reveal elastic material
28
Silver impregnation
shows reticular fibers and basement membranes
29
Sudan stains
- fat-soluble
- demonstrates lipids
- preservation of lipids cannot utilize alcohol so usually frozen specimens
30
Basic dyes
- react with anionic groups such as phosphate, sulfate, carboxyl groups
- nature of binding depends on pH
- tissues that react are called basophilic
31
Examples of basic dyes
- methyl green
- methylene blue
- pyronin G
- toluidine blue
- paramecium: fast green pH 2.5, shows trichosis
32
Acid dyes characteristics
- binds by forming electrostatic linkages with cationic groups such as amino groups of proteins
- acidophilic
- different sequences of acid dyes give different results
33
Mallory's triple stain
uses three acid dyes:
- aniline blue stains collagen
- acid fuchsin stains ordinary cytoplasm
- orange G stains RBCs
34
Examples of acid dyes
- acid fuchsin
- aniline blue
- eosin
- orange G
35
Metachromasia
phenomenon whereby a dye changes color after reacting with a tissue component
toluidine blue used to stain cartilage ground substance or mast cell granules
36
TEM tissue staining
- utilizes ions of heavy metals that are very electron-dense (heavy metals)
- heavy metals can be added during fixation, dehydration, or soaking in ionic solution after sectioning
37
Examples of TEM tissue stains
- osmium tetroxide
- uranyl nitrate
- uranyl acetate and lead
- For SEM, platinum or gold
38
Perls' reaction purpose and procedure
- used to demonstrate the presence of iron in tissue especially for patients with diseases that store iron (hemochromatosis)
- incubate tissues in a mixture of potassium ferrocyanide and HCl
- results are an insoluble blue precipitate of ferric ferrocyanide
39
Stains for lipids
- lipids are soluble in the reagents that are used in the normal processing of tissues
- frozen sections
- Sudan IV, Sudan black, oil red O, Nile blue
40
Schiff reagent reactions
- depends on formation of aldehyde groups following exposure to HCl or periodic acid
- Fuelgen reaction
- Periodic acid-Schiff reaction
41
Fuelgen reaction
- type of Schiff reagent reaction
- mild hydrolysis of HCl exposes aldehyde groups on deoxyribose
- reacts with aldehyde groups and forms a deep-pinkish color
- reacts with DNA (obvi)
42
Periodic acid-Schiff reaction (PAS)
- type of Schiff reagent reaction
- periodic acid is used to cleave bonds between adjacent carbons of carbohydrates and from an aldehyde group
- schiff reagent reacts with aldehyde groups
- PAS substances: polysaccharides, glycosaminoglycans, proteoglycans, glycoproteins, glycolipids
- clinical application: biopsies of tissues from patients with glycogenoses (glycogen storage diseases)
43
Best carmine
- instead of PAS, carbohydrate stain
| - demonstrates glycogen deposits
44
RNA stains
- basic dyes
- toluidine blue, methylene blue, methyl green
- control slides are necessary to distinguish other basophilic substances
- control slides are incubated with ribonuclease
45
Immunocytochemical techniques
Study the presence of tissue constituents (antigens: proteins, glycoproteins, proteoglycans) by using monoclonal antibodies
46
Antibodies used in immunocytochemical techniques
- monoclonal antibodies are derived from activated B cell clones exposed to a specific antigen- very specific
- most antigens have a variety of binding sites (epitopes) that generate a number of different antibodies (polyclonal)
- B lymphocytes can mutate into tumor cells resulting in myeloma
47
hybridoma
- fusion of a single activated B cell and myeloma cell
- grows indefinitely in culture
- produces a specific monoclonal antibody
48
Direct labeling using antibodies in immunocytochemical techniques
antibodies can be conjugated with
- fluorescent dye to produce a visible marker with fluorescent microscopy
- visible substance to produce visible marker for light microscopy
- gold or ferritin to produce a marker visible with electron microscopy
49
Indirect labeling of antibodies in immunocytochemical techniques
- marker is attached to a second antibody that is specific to the antibody used to locate the antigen of interest
- more commonly used
- produce a small number of kinds of secondary antibodies that can recognize a large number of primary antibodies (label secondary and cut your cost)
50
Examples of antibodies used as markers
anti-insulin slide: antibodies directed against insulin (beta cells on perimeter of islet)
anti-glucagon: antibodies directed against glucagon (alpha cells more centered on slide)
anti-IgG: actually labeling immune response in tonsil
