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Histology > Ex 1 TIssue Prep and Staining > Flashcards

Ex 1 TIssue Prep and Staining Flashcards

1
Q

steps of tissue prep

A
  • fixing
  • dehydration
  • removal of alcohol
  • embedding
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2
Q

steps of tissue prep

A
  • fixing
  • dehydration
  • removal of alcohol
  • embedding
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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

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4
Q

What are the characteristics of the ideal fixative?

A

give greater optical contrast (with staining) with the least amount of distortion

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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
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6
Q

What do acid fixatives fix?

A

chromatin, nucleoli, spindle fibers

do not fix mitochondria or nucleoplasm

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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
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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
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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
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10
Q

Basic fixatives

A
  • fix tissues where mitochondrial staining is desired

- chromatin is dissolved

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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
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12
Q

List of acid fixatives

A

Carnoy’s fluid
Zenker’s fluid
Bouin’s fluid

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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
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14
Q

Purpose of dehydration

A

Remove all water from tissue because it will be embedded and infiltrated with a hydrophobic material

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15
Q

How to complete dehydration

A

place tissue in successively increasing strengths of ethanol

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16
Q

Limitations of using ethanol, n-butyl alcohol, or acetone for dehydration?

A

dissolve neutral fats

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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.

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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

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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

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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)

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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
Q

Examples of stains

A
  • H&E (hematoxylin and eosin)
  • orcein and resorcin fuchsin stains
  • silver
  • Sudans
  • basic dyes
  • acid dyes
26
Q

H&E stains

A
  • 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
Q

Orcein and resorcin

A

reveal elastic material

28
Q

Silver impregnation

A

shows reticular fibers and basement membranes

29
Q

Sudan stains

A
  • fat-soluble
  • demonstrates lipids
  • preservation of lipids cannot utilize alcohol so usually frozen specimens
30
Q

Basic dyes

A
  • react with anionic groups such as phosphate, sulfate, carboxyl groups
  • nature of binding depends on pH
  • tissues that react are called basophilic
31
Q

Examples of basic dyes

A
  • methyl green
  • methylene blue
  • pyronin G
  • toluidine blue
  • paramecium: fast green pH 2.5, shows trichosis
32
Q

Acid dyes characteristics

A
  • binds by forming electrostatic linkages with cationic groups such as amino groups of proteins
  • acidophilic
  • different sequences of acid dyes give different results
33
Q

Mallory’s triple stain

A

uses three acid dyes:

  • aniline blue stains collagen
  • acid fuchsin stains ordinary cytoplasm
  • orange G stains RBCs
34
Q

Examples of acid dyes

A
  • acid fuchsin
  • aniline blue
  • eosin
  • orange G
35
Q

Metachromasia

A

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
Q

TEM tissue staining

A
  • 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
Q

Examples of TEM tissue stains

A
  • osmium tetroxide
  • uranyl nitrate
  • uranyl acetate and lead
  • For SEM, platinum or gold
38
Q

Perls’ reaction purpose and procedure

A
  • 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
Q

Stains for lipids

A
  • 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
Q

Schiff reagent reactions

A
  • depends on formation of aldehyde groups following exposure to HCl or periodic acid
  • Fuelgen reaction
  • Periodic acid-Schiff reaction
41
Q

Fuelgen reaction

A
  • 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
Q

Periodic acid-Schiff reaction (PAS)

A
  • 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
Q

Best carmine

A
  • instead of PAS, carbohydrate stain

- demonstrates glycogen deposits

44
Q

RNA stains

A
  • basic dyes
  • toluidine blue, methylene blue, methyl green
  • control slides are necessary to distinguish other basophilic substances
  • control slides are incubated with ribonuclease
45
Q

Immunocytochemical techniques

A

Study the presence of tissue constituents (antigens: proteins, glycoproteins, proteoglycans) by using monoclonal antibodies

46
Q

Antibodies used in immunocytochemical techniques

A
  • 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
Q

hybridoma

A
  • fusion of a single activated B cell and myeloma cell
  • grows indefinitely in culture
  • produces a specific monoclonal antibody
48
Q

Direct labeling using antibodies in immunocytochemical techniques

A

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
Q

Indirect labeling of antibodies in immunocytochemical techniques

A
  • 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
Q

Examples of antibodies used as markers

A

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

Histology (7 decks)

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