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Histology Spring 2020 > Chapter 8: Connective Tissue > Flashcards

Chapter 8: Connective Tissue Flashcards

1
Q

Masson’s Trichrome Purpose

A

To differentiate between Collagen and smooth muscle

esp for tumors and identifying increases in collagenous tissue for diseases such as cirrhosis of the liver

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2
Q

Masson’s Trichrome Principle

A

Uses 3 dyes:
Biebrich Scarlet: acid dye that stains all acidophilic tissue red (cytoplasm, muscle, and collagen)
Phosphotungstic Acid PTA or Phosphomolybdic Acid PMA: This acid causes the scarlet to diffuse out of collagen, which is more permeable than cytoplasm, which remains red in addition to muscle fibers
Aniline Blue: stains collagen blue by binding to the PTA/tissue complex (PTA is like the primary and blue is like the secondary)

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3
Q

Masson’s Trichrome Preferred Fixative

A

Bouin’s is preferred, 10% NBF may be used

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4
Q

Masson’s Trichrome Basic Procedure

A
  1. Deparaffinize and hydrate
  2. Rinse in water
  3. Mordant in Bouin’s
  4. Wash in running water to remove yellow from Bouin’s
  5. Rinse in water
  6. Stain in Weigert Iron Hematoxylin
  7. Wash in running water
  8. Rinse in water
  9. Stain in Biebrich Scarlet Acid-Fuchsin
  10. Rinse in water
  11. PTA/PMA to differentiate collagen from cytoplasm and muscle
  12. Stain in aniline blue
  13. Rinse in water
  14. Differentiate collagen in 1% acetic acid
  15. Dehydrate, clear, coverslip
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5
Q

Masson’s Trichrome Results

A

Nuclei: black
Cytoplasm, keratin, muscle fibers: red
collagen and mucin: blue

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6
Q

Masson’s Trichrome Technical Notes

A

Light green is an alternative counterstain to aniline blue, especially when collagen is predominant

pale blue collagen staining is a sign of overdifferentiation with acetic acid

Picric acid is explosive when less than 10% aqueous, make sure it (the Bouin’s) doesn’t spill and evaporate in the oven during heating step

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7
Q

Gomori 1-Step Trichrome Purpose

A

Differentiation between collagen and smooth muscle fibers

Also for identifying an increase in collagenous connective tissue fibers

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8
Q

Gomori Trichrome Principle

A

Uses one stain made of three components to reduce the number of steps

Chromotope 2R stains plasma red
Fast green FCF, light green, or aniline blue stains collagen

These two stains are combined with PTA and acetic acid, which causes muscle and cytoplasm to stain red

The tungstate ion binds to collagen, and that metal/tissue complex binds the aniline blue collagen stain, which is then differentiated by acetic acid

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9
Q

Gomori Trichrome Preferred Fixative

A

Any except Bouin’s (which is used as a mordant to intensify the stain colors)

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10
Q

Gomori Trichrome Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Rinse in water
  3. Mordant in Bouin’s
  4. Wash in running water
  5. Stain in Gomori Trichrome stain
  6. Differentiate in 0.5% Acetic Acid
  7. Dehydrate, clear, coverslip
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11
Q

Gomori Trichrome Results

A

Nuclei: black
Cytoplasm, keratin, muscle fibers: red
Collagen and mucin; blue or green depending on the counterstain

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12
Q

Gomori Trichrome Technical Notes

A

Color intensity can be varied by changing the pH
pH 1.3 gives the best binding, but acetic acid is pH2.5
1.3 can be obtained by using HCl

Zinc-formalin is a good fixative for trichrome stain that doesn’t require mordanting with Bouin’s

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13
Q

Van Gieson (Picric Acid-Acid Fuchsin) Purpose

A

Usually used as a counterstain rather than a primary stain, especially for elastic techniques such as Verhoeff-van Gieson VVG or elastic-van Gieson EVG

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14
Q

Van Gieson (Picric Acid-Acid Fuchsin) Principle

A

In a strong acidic solution, collagen is selectively stained by acid-fuchsin, which is an acid aniline dye
HCl differentiates between collagen and muscle fibers
Picric acid provides acidic pH and stains the muscle and cytoplasm

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15
Q

Van Gieson (Picric Acid-Acid Fuchsin) Preferred Fixative

A

Any

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16
Q

Van Gieson (Picric Acid-Acid Fuchsin) Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in Weigert Iron Hematoxylin
  3. Wash in running water
  4. Stain in Van Gieson (acid fuchsin+picric acid)
  5. 95% alcohol
  6. Dehydrate, clear, coverslip
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17
Q

Van Gieson (Picric Acid-Acid Fuchsin) Results

A

Nuclei: black
Collagen: Brilliant red
Muscle and Cytoplasm: yellow

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18
Q

Van Gieson (Picric Acid-Acid Fuchsin) Technical Notes

A

Weigert Iron hematoxylin resists acidic solutions

pH of picric acid is very important, if the solution is not saturated the collagen cytoplasm and muscle fibers may all stain pale pink to pale orange
HCl can help to sharpen color differentiation

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19
Q

What are the 3 types of connective tissue fibers?

A

Elastic, Collagen, Reticular

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20
Q

Elastic

A

Most abundant in tissue requiring flexibility, because they allow tissue to stretch
Present in most fibrous connective tissue
not seen on HandE

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21
Q

Collagen

A

Provide strength to connective tissue proper
Very eosinophilic
Seen under light microscopy
Birefringent under polarized light
Cross-striated in EM
Demonstrated with Masson’s or Gomori Trichrome stains

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22
Q

Reticular

A

Type of Collagen
Not seen in HandE
Absorb silver from solutions
Smaller than most collagen fibers

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23
Q

What are the three types of muscle tissue?

A

Skeletal, smooth, and cardiac

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24
Q

Skeletal muscle type 1 (slow twitch)

A

.

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25
Q

Skeletal muscle type 2 (fast twitch)

A

.

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26
Q

Smooth Muscle

A

No striations, no branching, looks a lot like connective tissue on H and E, central mono-nucleated, involuntary, different ratio of actin and myosin than skeletal

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27
Q

Skeletal Muscle

A

Striations, no branching, Z-lines, peripheral multinucleated, voluntary
Actin and myosin are the major contractile proteins

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28
Q

Cardiac Muscle

A

Striations, branching, intercalated disks, mono-nucleated, involuntary

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29
Q

What are the 7 types of connective tissue cells?

A 
Fibroblasts
Mesenchymal Cells
Adipose
Mast Cells
Macrophages
Plasma Cells
Blood Cells
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30
Q

Fibroblasts

A

Main cell type and most common in loose CT
Almost the only cell type in regular CT
Flattened nuclei, spindle shaped cells

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31
Q

Mesenchymal Cells

A

Primitive, relatively undifferentiated
Look very similar to fibroblasts
Differentiate when needed

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32
Q

Adipose

A

Fat cells which synthesize and store lipids
Common in loose connective tissue
Flattened nucleus
Must be cut frozen at -30C because paraffin processing does not preserve lipids

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33
Q

Mast Cells

A

Trigger immune and inflammatory responses
Abundant secretory granules
Contain Histamine and Heparin
Exhibit metachromasia when stained with Toluidine Blue
Resemble basophilic leukocytes found in the blood

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34
Q

Macrophages

A

“Big eaters”
Scavenger cells that process antigenic material and present it to the lymphocytes
Blood leukocytes are their precursors

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35
Q

Plasma Cells

A

Derived from B lymphocytes
Produce immunoglobulins
Deeply basophilic cytoplasm

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36
Q

Blood Cells

A

Many types

Red blood cells are the most common

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37
Q

What is the basement membrane?

A

Also called the basal lamina
Between the epithelium and underlying connective tissue
Demonstrated by carbohydrate methods because they contain more sugar than ordinary collagen

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38
Q

What is the function of the basement membrane?

A

Beneath the epithelium and connects it to the underlying connective tissue
Provides physical support to the epithelium, cell attachment, and ultrafiltration

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39
Q

What is a unique feature of mast cells that helps in demonstration?

A

Exhibit metachromasia when stained with Toluidine Blue

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40
Q

Which procedures are used to demonstrate lipids?

A

Oil Red O, Sudan Black, Toluidine Blue

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41
Q

Describe Silver Oxidation

A

Oxidizes reticulin to aldehyde groups

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42
Q

Describe Silver Sensitization

A

Deposit of metallic salts near but not on the reticulin, this metal is later replaced by silver during impregnation

43
Q

Describe Silver Impregnation

A

Tissue is treated with ammoniacal silver to deposit silver on the reticulin,

44
Q

Describe Silver Reduction

A

The aldehyde groups in the reticulin reduce the diamine silver to metallic silver

45
Q

Describe Silver Toning

A

Bound metallic silver is treated with Gold chloride, changing the fibers from brown to black

46
Q

Movat Pentachrome Purpose

A

Demonstration of mucin, fibrin, elastic fibers, muscle, and collagen

47
Q

Movat Pentachrome Principle

A
  1. Acidic mucosubstances are stained by Alcian blue
  2. Alkaline alcohol converts Alcian blue to monastral fast blue which is insoluble (this prevents decolorization in later steps)
  3. Iron hematoxylin stains the elastic fibers which are then diffed with ferric chloride
  4. Sodium Thiosulfate removes excess iodine from the iron hematoxylin
  5. Crocein scarlet and acid fuchsin are acid dyes that stain muscle, cytoplasm, collagen, and ground substance red
  6. PTA (phosphotungstic acid) differentiation removes stain from collagen and ground substance
  7. Acetic Acid removes PTA
  8. Collagen is counterstained with Alcoholic safran (yellow)
48
Q

Movat Pentachrome Preferred Fixative

A

10% NBF

49
Q

Movat Pentachrome Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in alcian blue
  3. Wash in running water
  4. “Fix” Alcian blue to monastral using alkaline alcohol
  5. Wash thoroughly in running water
  6. Rinse in distilled water
  7. Stain in Iron hematoxylin
  8. Rinse in water
  9. Differentiate in 2% ferric chloride until black elastic fibers contrast sharply against background
  10. Rinse in water
  11. Sodium Thiosulfate to remove iodine
  12. Wash/rinse in water
  13. Stain in Crocein scarlet-acid fuchsin
  14. Rinse in water
  15. Rinse in 0.5% Acetic Acid
  16. Place in 5% PTA (decolorizes collagen and ground substance)
  17. Rinse in 0.5% Acetic Acid
  18. Rinse in Absolute Alcohol
  19. Stain in alcoholic safran (yellow)
  20. Rinse in Absolute alcohol
  21. Clear, coverslip
50
Q

Movat Pentachrome Results

A 
Nuclei and elastic fibers: Black
Collagen: Yellow
Ground Substance and mucin: blue (or green)
Fibrinoid, fibrin: Intense Red
Muscle: Red (less vibrant)
51
Q

Movat Pentachrome Technical Notes

A

Differentiation of elastic fibers takes 2-3 minutes
Very important to completely remove the alkaline alcohol with running water, otherwise subsequent steps will be inhibited
Incidentally can demonstrate cryptococcus neoformans bright blue

52
Q

Verhoeff Van Gieson Purpose

A

To demonstrate pathologic changes in elastic fibers; such as atrophy, thinning, loss, breaks, or splitting that may result from vascular diseases

53
Q

Verhoeff Van Gieson Principle

A
  1. Tissue is overstained with hematoxylin-ferric chloride-iodine lake (the metals act as both mordants and oxidizers)
  2. Excess ferric chloride mordant acts as a differentiator by breaking the tissue-mordant-dye complex
  3. Because elastic tissue has high affinity for the iron-hematoxylin, other tissues are decolorized during differentiation
  4. Sodium Thiosulfate removes excess Iodine
  5. Van Gieson acts as Counterstain
54
Q

Verhoeff Van Gieson Preferred Fixative

A

Any, but 10% NBF is preferred

55
Q

Verhoeff Van Gieson Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in Verhoeff elastic tissue stain (alcoholic hematoxylin, ferric chloride, Lugol iodine)
  3. Wash in water
  4. Differentiate (1 slide at a time) in ferric chloride until elastic fibers are distinct and background is colorless to light grey
  5. Rinse in Water
  6. Sodium Thiosulfate (to remove iodine, also differentiates a little because of picric acid component)
  7. Wash in running tap water
  8. Counterstain in Van Gieson stain
  9. Differentiate in 95% alcohol
  10. Dehydrate, clear, and coverslip
56
Q

Verhoeff Van Gieson Results

A

Elastic Fibers: Blue-black to black
Nuclei:Blue to black
Collagen: Red
Other tissue elements: Yellow

57
Q

Verhoeff Van Gieson Technical Notes

A

Easy to overdiff, better to underdiff, and you can always re-stain as long as you haven’t reached the alcohol step

Don’t use mercuric fixatives because they are toxic

Slides must be individually differentiated depending on the amount of elastic tissue present

58
Q

Gomori Reticulin Purpose

A

Demonstrate reticular fibers, used to diagnose some liver diseases and tumors

59
Q

Gomori Reticulin Principle

A

Oxidizer: potassium permanganate to form aldehydes, excess removed by potassium metabisulfate
Sensitizer: ferric ammonium sulfate to “prep” tissue
Impregnation solution: ammoniacal silver (diamine silver)
Reducing agent: Formalin
Toner: gold chloride (brown to black color change)
Sodium thiosulfate to remove excess silver

60
Q

Gomori Reticulin Preferred Fixative

A

10% NBF

61
Q

Gomori Reticulin Basic Procedure

A
  1. Deparaffinize, hydrate
  2. oxidize in potassium permanganate
  3. Rinse in tap water
  4. Differentiate in potassium metabisulfite
  5. Wash in tap water
  6. Sensitize in ferric ammonium sulfate
  7. wash in tap water, then distilled
  8. Impregnate with ammoniacal silver solution
  9. Rinse in distilled water
  10. Reduce in 20% formalin
  11. Wash in tap water
  12. Tone in gold chloride
  13. Rinse in distilled water
  14. Treat with potassium metabisulfite
  15. Treat with Sodium thiosulfate to remove unreacted silver
  16. Wash in tap water
  17. Counterstain with nuclear fast red
  18. Wash thoroughly to prevent cloudiness
  19. Dehydrate, clear, coverslip
62
Q

Gomori Reticulin Results

A

Reticulin: black
Collagen: taupe
Other tissue elements: pink-red if NFR is used as a counterstain

63
Q

Gomori Reticulin Technical Notes

A

Don’t over saturate silver with ammonia because it reduces sensitivity of the stain

need clean glassware

silver should deposit linearly not granularly

Store silver solutions in fridge to prevent formation of explosive ammonia compounds

64
Q

Gordon and Sweets Reticulin Purpose

A

Demonstrate reticular fibers, used to diagnose some liver diseases and tumors

65
Q

Gordon and Sweets Reticulin Principle

A 
Oxidizer: potassium permanganate
Sensitizer: ferric ammonium sulfate
Impregnator: ammoniacal silver
Reducer Formalin
Toner: gold chloride
66
Q

Gordon and Sweets Reticulin Preferred Fixative

A

10% NBF

67
Q

Gordon and Sweets Reticulin Basic Procedure

A
  1. Deparaffinize, hydrate
  2. oxidize in potassium permanganate
  3. Rinse in tap water
  4. Bleach in 1% oxalic acid
  5. Wash in tap water
  6. Sensitize in ferric ammonium sulfate
  7. wash in distilled water
  8. Impregnate with ammoniacal silver solution
  9. Rinse in distilled water
  10. Reduce in 10% formalin
  11. Wash in tap water
  12. Tone in gold chloride
  13. Wash in tap water
  14. Treat with Sodium thiosulfate to remove unreacted silver
  15. Rinse in distilled water
  16. Counterstain with nuclear fast red
  17. Wash thoroughly in distilled water to prevent cloudiness
  18. Dehydrate, clear, coverslip
68
Q

Gordon and Sweets Reticulin Results

A

Reticulin: black

Other tissue elements: pink-red if nuclear fast red is used as counterstain

69
Q

Gordon and Sweets Reticulin Technical Notes

A

Less background than gomori

Don’t over saturate silver with ammonia because it reduces sensitivity of the stain

need clean glassware

silver should deposit linearly not granularly

Store silver solutions in fridge to prevent formation of explosive ammonia compounds

70
Q

Mallory PTAH Purpose

A

Demonstrate muscle cross-striations and fibrin (also glial fibers and myelin); these cross-striations can be found in rhabdomyosarcomas (muscle cell tumors)

71
Q

Mallory PTAH Principle

A

High ratio of PTA to hematein results in a blue lake that stains cross striations and fibrin blue while excess PTA stains collagen red-brown

72
Q

Mallory PTAH Preferred Fixative

A

Zenker preferred, 10%NBF acceptable

73
Q

Mallory PTAH Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Mordant in Zenker overnight if fixed in 10%NBF
  3. Rinse in tap water
  4. Place in Gram Iodine
  5. Rinse in tap water
  6. Place in 5% Sodium Thiosulfate
  7. Wash in tap water
  8. Place in potassium permanganate (oxidizer)
  9. Rinse in tap water
  10. Place in 5% oxalic acid (bleacher)
  11. wash in running tap water
  12. stain in PTAH solution overnight
  13. Dehydrate, clear, coverslip
74
Q

Mallory PTAH Results

A

Cross-striations, fibrin: blue
Nuclei: blue
Collagen: red-brown

75
Q

Mallory PTAH Technical Notes

A

PTAH that is chemically oxidized with potassium permanganate has a shorter half-life than naturally ripened PTAH, solution should be stored in amber glass bottles to slow over oxidation

Tissue needs to be well hydrated before staining so the tissue structures can take up the dye molecules

Sodium thiosulfate can interfere with PTAH binding, so washing must be very thorough

Bouin can be used as a mordant

NBF fixed tissue tends to stain unevenly, even with mordanting

76
Q

Oil Red O Purpose

A

Demonstrate neutral lipids in frozen sections

Can help show fatty emboli due to bone fracture or liposarcomas

77
Q

Oil Red O Principle

A

Physical method of staining
The dye is more soluble in tissue lipid than the solvent it is dissolved in
The dye cannot be water soluble
Must be strongly colored
Isopropanol and propylene glycol are preferred solvents

78
Q

Oil Red O Preferred Fixative

A

10%NBF or calcium-formal

No alcohols, they dissolve lipids

79
Q

Oil Red O Basic Procedure

A
  1. Cut frozen sections, fix in 40% formaldehyde, wash well, blot
  2. stain in oil red O
  3. wash in tap water
  4. Stain in Harris Hematoxylin containing acetic acid
  5. wash in tap water
  6. Blue in ammonia water
  7. Wash in tap water
  8. Mount with aqueous mounting media
  9. Seal the edges of the coverslip with nail polish
80
Q

Oil Red O Results

A

Fat: Red

Other tissue elements: depends on method used

81
Q

Oil Red O Technical Notes

A

Do not process for paraffin embedding because alcohol dissolves lipid

Formalin fixed tissue infiltrated with 30% sucrose before freezing improves frozen sectioning

Must use aqueous mounting media

Don’t push on the coverslip or the fat will be displaced

Make sure to filter the oil red O stain

82
Q

Sudan Black B Purpose

A

To demonstrate neutral lipids, most sensitive of the lipid dyes and more soluble in phospholipids than oil red O

Used in hematopathology to differentiate granulocyte precursors from leukocytes

83
Q

Sudan Black B Principle

A

Soluble in neutral fats, is slightly basic which allows it to combine with acidic groups in compound lipids like phospholipids

84
Q

Sudan Black B Preferred Fixative

A

10% NBF or post-fixed in calcium-formalin

NO ALCOHOL

85
Q

Sudan Black B Basic Procedure

A
  1. Place fixed and rinsed frozen sections in propylene glycol
  2. Stain in Sudan Black B
  3. Differentiate in propylene glycol
  4. Wash in distilled water
  5. Counterstain with Nuclear Fast Red
  6. Wash well in distilled water (to prevent cloudiness)
  7. Mount with aqueous mounting media
86
Q

Sudan Black B Results

A

Fat: Blue-black
Nuclei: Red

87
Q

Osmium Tetroxide Purpose

A

Demonstrate fat in a way that allows paraffin embedding

88
Q

Osmium Tetroxide Principle

A

Osmium tetroxide chemically combines with fat, blackening it in the process
This is the only chemical method for fat because osmium tetroxide is insoluble in alcohol and xylene

89
Q

Osmium Tetroxide Preferred Fixative

A

10%NBF initially, then trim to 2mm thick for osmium tetroxide penetration

90
Q

Osmium Tetroxide Basic Procedure

A
  1. Trim 10%NBF fixed tissue to 2mm thick, wash in running tap water
  2. Rinse in distilled water
  3. Place tissue in osmium tetroxide under the hood
  4. Rinse in distilled water
  5. Differentiate the tissue in periodic acid, background will clear leaving fat black
  6. Wash in running tap water
  7. Process routinely, starting with 70% alcohol, embed as usual
  8. Cut paraffin sections at 4-5uM
  9. Deparaffinize and hydrate as usual
  10. Stain with routine HandE, or any desired special stain
  11. Dehydrate, clear, coverslip
91
Q

Osmium Tetroxide Results

A

Fat: black

Other tissue elements: according to method used

92
Q

Osmium Tetroxide Technical Notes

A

Gross fat won’t be fixed, but small lipid droplets are well demonstrated

face carefully and section asap because osmium tetroxide has poor penetration in tissue

cytoplasm will be gray and will effect quality of cytoplasmic stains

93
Q

Toluidine Blue Purpose

A

Demonstrate mast cells in tissue

Which play a key role in inflammation, allergic reactions, autoimmune disorders, and mastocytomas

94
Q

Toluidine Blue Principle

A

Mast cells stain metachromatically (different color than the dye) Look purple with blue nuclei against a blue background
Dependent on pH, dye concentration, and temperature

95
Q

Toluidine Blue Preferred Fixative

A

10%NBF?

96
Q

Toluidine Blue Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in toluidine blue
  3. Rinse in distilled water
  4. Dehydrate, clear, coverslip
97
Q

Toluidine Blue Results

A

Mast cells: deep rose-violet

Background: blue

98
Q

Toluidine Blue Technical Notes

A

Alcohol is ok in this procedure because the mast cell granules maintain their metachromatic staining

This is a rapid screening method for mast cells

Mast cells will also stain orange-red with MGP due to high amount of RNA

99
Q

Aldehyde Fuschin Stain Purpose

A

To demonstrate pathologic changes in elastic fibers; such as atrophy, thinning, loss, breaks, or splitting that may result from vascular diseases
(Same as Verhoeff)

100
Q

Aldehyde Fuschin Stain Principle

A

HCl and paraldehyde are added to an alcoholic solution of basic fuchsin to form aldehyde fuchsin
Schiff bases are formed by the aldehyde and the fuchsin

101
Q

Aldehyde Fuschin Stain Preferred Fixative

A

10% NBF, no chromate (B5, Zenker, Helly)

102
Q

Aldehyde Fuschin Stain Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in aldehyde fuchsin solution
  3. Rinse off excess stain with 70% Alcohol
  4. Wash in water and check for staining of elastic fibers. Further diff in 70% alcohol if necessary
  5. Rinse in water
  6. Counterstain with Light Green
  7. Dehydrate, clear, coverslip
103
Q

Aldehyde Fuschin Stain Results

A

Elastic Fibers: Deep blue to deep purple

Other tissue elements: Green

104
Q

Aldehyde Fuschin Stain Technical Notes

A

Paraldehyde needs to be freshly made
Acetaldehyde can be used instead and is good for 3 weeks
Old solutions of aldehyde fuchsin age like Schiff’s and loose strength over time

Histology Spring 2020 (14 decks)

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