Lecture 3

Question 1

What steps must be tkaen to prepare a tissue for observation?

Answer 1

• Fixing
• Dehdydration
• Removal of Alcohol
• Embedding

Question 2

Fixing

Answer 2

• Fixing prevents further deterioration of the tissue specimen and helps to harden the tissue prior to embeddign and sectioning
• Any fixative , however,radically distorts the specimen
• The ideal fixatives give the greater optical contrast(with staining) with the least amount of distortion
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Question 3

Formalin

Answer 3

• One of the most widely used fixing agents
  
  • May be used alone (often in a buffered 10% solution) or in combination with other agents scuh as alcohol(shrinks tissues) and/or acetic acid(softens and counteracts the shrinkage of alcohol)
  • Reacts with amino acids of the tissue proteins and stabilizes tissue stcuture to prevent further deterioration
  • Not god if fine cytological detail is desired
• Good for growth fixation
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Question 4

Acid Fixatives

Answer 4

• Fix chromatin,nucleoli, and spindle fibers but not mitochondria or nucleoplasm
  
  • Carnoy’s fluid is a mixture of alcohol, chloroform, and glacial acetic acid.It is a good general fixative and is useful for preserving glycogen in animal tissues
  • Zenker’s fluid contains potassium dichromate, mercuric chloride ,and glacial acetic acid. It is useful when sharp histological detail is desired but must be washed out carefully to prevent the precipitation of black crystals
  • Bouin’s fluid contains picric aid,formalin, and glacial acetic acid. It is a widely used general fixative that gives good cytological detail.It requires a prolonged and careful washing cycles
• Picric acid if you don’t wash your tisseus will be yellow , and the other problem is it’s highly explosive in crystalline form

Question 5

Basic Fixative

Answer 5

• Basic fixatives can be ued to fix tissues where mitochondrial staining is desired. In this fixing proceudre, chromatin is dissolved
  
  • Zirkle-Erliki fixative contains potassium dichromate,ammonium dichromate,copper sulfate , and distilled water
  • It requires a long fixing time(2 days) and washing under running water
• Fixatives for TEM:glutaraldehyde
  
  • perserves proteins by cross linking them
• Osmium tetroxide
  
  • Reacts with lipids(esp. Phospholipids) and imparts electron density to cell and tisseus structures

Question 6

Dehydrating

Answer 6

• Because the tissue sample will eventually be embedded and infiltrated with a hydrophobic material(usually parafiin), all the water must be removed from the tissue.
  
  • Dehydration consists of placing the tissue in succesively increasing strengths of ethanol until all the water is removed
  • Ethanol dissolves neutral fats and cannot be used for dehydration if it is desirable to leave these intact
  • N-butyl alcohol or acetone may also be used for dehydration
• When you mix alcohol and water, creates eddy currents which happens in the tissue
  
  • So thats why it’s a slow progressive process
• If you want to perserve fats, freeze the tissue and use a freezing microphone to cut the tissue

Question 7

Clearing

Answer 7

• Consists of replacing the alcohol with an agent such as xylene or cedar oil
  
  • note that the paraffin embedding medium will not mix with alcohol but will mix with xylene or other clearing agents
  • Other clearing agents include cedar oil and carbon tetrachloride

Question 8

Embedding

Answer 8

• Tissue specimen is moved sequentially through several (Usually three) melted paraffin baths
• After the final bath the specimen is placed in a mold that is then filled with melted paraffin
• The paraffin mold is rapidly hardened by placing it in a cold water bath

Question 9

Embedding for TEM

Answer 9

• Tissues are infiltrated with a monomeric resin(epoxy resin)
• Resin is then polymerized
• tissue samples are typically less than 1mm^3

Question 10

Tissue sectioning

Answer 10

• Sectioning is typically done a rotary microtome which utilizes a very sharp blade over which the paraffin block is raised and lowered after being advanced a fixed distance per cycle
• Sectioning can also be done using a sharp razor and a tubular holder in which the speciemn is tightly held
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Question 11

Sectioning for TEM

Answer 11

• Sections are cut a 50 to 150nm
• Diamond knives are used (stainless steel not sharp enough)
• sections are too fragile normal handling and must be floated onto a plastic coated copper mesh gri
  
  • The wholes in the grid are ver smally , but from a TEM perspecitve the holes are huge
• Plastic is left in place during viewing
  
  • Thee to help support the fragile specimen
  • Holes in copper plate allow the TEM electrons to pass throuhg
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Question 12

Animal tissues

Answer 12

Typically colorless’they must be artfically colored(stained) to bring out detail

Question 13

How do you prepare an animal tissue for staining?

Answer 13

• The paraffin must be removed from the section , which is now mounted on a microscope slide
• Accomplished with xylene
• xylene must be removed using a graded series of alcohol down to water
• Stains are then applied and the section is again dehydrated through a graded series of alcohols
• Alcohol is removed with xylene
• Drop of cement followed by a cover slip is applied

Question 14

Which stains are used to help display strucutural features?

Answer 14

• Hematoxylin and eosin

Question 15

Hematoxylin

Answer 15

• Several kinds of hematoxylin preparations that stain nuclear material and some cytoplasmic components such as RER , dark blue to light blue or purple
• Although not a basic dye , hematoxylin behaves like on due to the properties of the mordant that is used to help it bind to the tissues
• Derived from longwood as hematein
• mordant has to be added as a seperate process , and some hematoxylin dyes need addition of mordant some already have it built in

Question 16

Eosin

Answer 16

• An acid dye stains most of they cytoplasmic components and much of the extracellular material a yellowish to pinkish color

Question 17

What stains can be used specifically to reveal elastic material?

Answer 17

orcein and resorcin fuchsin stains

Question 18

Which stain is useful to show reticular fibers and basement membranes?

Answer 18

Silver impregnation

Question 19

How can lipids be stained?

Answer 19

LIpids can be demontrated using fat soluble stains such as the Sudans, but preservation of lipids requirs techniques that do not utilize alcohol for dehydration,etc.

Question 20

Basic Dyes

Answer 20

• Basic dyes with the anionic groups of tissue components such as phosphate groups , sulfate groups and carboxyl groups
• Exact nature of binding depends on the pH. At a high pH all three groups are available for binding with the dye
• Any tissue components that reacts with a basic dye(or with hematoxylin) is said to be basophilic

Question 21

Examples of basic Dyes

Answer 21

• Methyl green
• Methylene blue
• Pyronine G
• Toluidine blue

Question 22

Acid Dyes:

Answer 22

• Acidic dyes bind to tissue components by forming electrostatic linkages with catioinic groups such as the amino groups of proteins
• Different types of acid dyes have slightyl different properties and can be used in sequence to give different results:
  
  • Mallory’s triple stain uses three acid dyes sequentially:aniline blue stains colalgen,acid fuchsin stains ordinary cytoplasm, and organge G stains red blood cells
• pH is goign to alter the chemical structre of the stain , and how light is refelcted from the specimen
• Any tissue component that reacts with an acid dye is said to be acidophilic

Question 23

Examples of Acid dyes:

Answer 23

• Acid Fuschsin
• Aniline Blue
• Eosin
• orange G

Question 24

Metachromasia

Answer 24

• This term refers to a phenomenon whereby dye chages color after reacting with a tissue component
• ex. toluidien blue used to stain cartilage ground substance or mast cell granules
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Question 25

How do you stain TEM

Answer 25

• Staing for TEM utilizes ion of hevay metals that are very electron dense
• Heavy metal can be added during fixation,dehydration, or by soaking in ionic solutions after sectioning
• Ex.
  
  • osmium tetroxide
  • uranyl nitrate
  • uranyl acetate and lead citrate
  • For SEM,platinum or gold may be used
• If they bind to the ion they are electron dense , and if not they are electron loose

Question 26

Histochemical staining Techniques

Answer 26

• Histochemical techniques can be used to study the chemistry of cells and tissues
• Ex.
  
  • Perl’s reaction
    
    • uesd to demonstrate the presence of iron in tissues , esp. in patients with diesase that store iron(ex hemochromatosis)
    • Incubate tisseus in a mixture of potassium ferrocyanide and HCL. Results are an insoluble blue precipitate of ferric ferrocyanide

Question 27

How would you stain lipids using histochemical techniques?

Answer 27

• Remember that lipids are soluble in the reagents that are used in the normal processing of tissues for histological examination
• Therefore , the use of frozen sections gives the best results when viewing lipids
• Use dyes that are soluble in lipids such as Sudan IV, Sudan black,oil red O, and Nile Blue?

Question 28

Schiff reagents reactions

Answer 28

• Reaction depends on the formation of aldehyde groups following exposure to HCl or periodic acid
• Will stain those particular aldehyde groups and can stain DNA

Question 29

Fuelgen reaction

Answer 29

• Mild hydrolysis with HCl exposes aldehyde groups on deoxyribose
• Schiff reagent reacs with the aldehyde groups and forms a deep pinkish color

Question 30

Periodic acid-Schiff reaction(PAS)

Answer 30

• Periodic acid is used to cleave bonds between adjacent carbons of carbohydrates and form aldehyde groups
• Schiff reagent reacts with the aldehyde groups and forms a deep pinkish color
• when you apply the schiff reagent you are going to be staining carbohydrate groups
• Will stain anything with carbohydrates in it

Question 31

PAS-positive substances:

Answer 31

• Polysacchariedes(glycogen)
• Glycosaminoglycans
• Proteoglycans
• Glycoproteins
• Glycolipids

Question 32

PAS clinical applications

Answer 32

• Biopsies of tisseus from patients with glycogenoses(glycogen storage diseases)
  
  • in liver: see definite deposits in the liver cell

Question 33

Best Carmine

Answer 33

• A dye that may also be sued to demonstrate glycogen deopsits
• part of histochemical staining techniques

Question 34

RNa stains

Answer 34

• RNA rich organelles may be stained with basic dyes
  
  • Toluidine blue
  • Methylene blue
  • Methyl green
• For this technique, control slides are necessary to distinguish other basophilic substances
  
  • Control slides are incubated with ribonuclease

Question 35

Immunocytochemical techniques

Answer 35

• Can be used to study the presence of specific tisseus constituents(antigens) by using monoclonal antibodies
• Antigens
  
  • Proteins,glycoproteins,proteoglycans
• Antibodies:
  
  • Monoclonal antibodies are derived from activated B cell clones exposed to a specific antigen. These antibodies are very specific
  • Most antigens have a variety of epitopes(binding sites) that generate a number of different antibodies- polyclonal
  • single immune response to an antigen is referred to as monoclonal
  • B lymphocyte can mutate into tumor cells resulting in a myeloma
  • Myeloma cells acquire the ability to grow indefinetly in culture
  • Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinetly in culture and produce a specific monoclonal antibody

Question 36

Direct labeling vs Indirect labeling related to antibodies in immunocytochemical techniques

Answer 36

• Direct labeling
  
  • Antibodies can be conjugated with a fluorescent dye to produce a visible marker with fluorescent microscopy
  • antibodies can be conjugated wtih a visible substance to produce a visible marker for light microscopy
  • Antibodies can be conjugated with gold or ferritin to prodce a marker visible with electron micrscopy
• Indirect labeling
  
  • In this variation , the marker is attached to a second antibody which is specific to the antibody used to locate the antigen of interest
• easiest way to see antibody is to add marker becasue you can’t see them without it
  
  • Secondary antibody treats the primary antibody as antigen
  • Primary antibodies are usually IGG and produce asecondary antibodies to IGG that will recognize IGG
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