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
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)
3
Q
Masson’s Trichrome Preferred Fixative
A
Bouin’s is preferred, 10% NBF may be used
4
Q
Masson’s Trichrome Basic Procedure
A
- Deparaffinize and hydrate
- Rinse in water
- Mordant in Bouin’s
- Wash in running water to remove yellow from Bouin’s
- Rinse in water
- Stain in Weigert Iron Hematoxylin
- Wash in running water
- Rinse in water
- Stain in Biebrich Scarlet Acid-Fuchsin
- Rinse in water
- PTA/PMA to differentiate collagen from cytoplasm and muscle
- Stain in aniline blue
- Rinse in water
- Differentiate collagen in 1% acetic acid
- Dehydrate, clear, coverslip
5
Q
Masson’s Trichrome Results
A
Nuclei: black
Cytoplasm, keratin, muscle fibers: red
collagen and mucin: blue
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
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
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
9
Q
Gomori Trichrome Preferred Fixative
A
Any except Bouin’s (which is used as a mordant to intensify the stain colors)
10
Q
Gomori Trichrome Basic Procedure
A
- Deparaffinize, hydrate
- Rinse in water
- Mordant in Bouin’s
- Wash in running water
- Stain in Gomori Trichrome stain
- Differentiate in 0.5% Acetic Acid
- Dehydrate, clear, coverslip
11
Q
Gomori Trichrome Results
A
Nuclei: black
Cytoplasm, keratin, muscle fibers: red
Collagen and mucin; blue or green depending on the counterstain
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
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
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
15
Q
Van Gieson (Picric Acid-Acid Fuchsin) Preferred Fixative
A
Any
16
Q
Van Gieson (Picric Acid-Acid Fuchsin) Basic Procedure
A
- Deparaffinize, hydrate
- Stain in Weigert Iron Hematoxylin
- Wash in running water
- Stain in Van Gieson (acid fuchsin+picric acid)
- 95% alcohol
- Dehydrate, clear, coverslip
17
Q
Van Gieson (Picric Acid-Acid Fuchsin) Results
A
Nuclei: black
Collagen: Brilliant red
Muscle and Cytoplasm: yellow
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
19
Q
What are the 3 types of connective tissue fibers?
A
Elastic, Collagen, Reticular
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
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
22
Q
Reticular
A
Type of Collagen
Not seen in HandE
Absorb silver from solutions
Smaller than most collagen fibers
23
Q
What are the three types of muscle tissue?
A
Skeletal, smooth, and cardiac
24
Q
Skeletal muscle type 1 (slow twitch)
A
.
25
Skeletal muscle type 2 (fast twitch)
.
26
Smooth Muscle
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
27
Skeletal Muscle
Striations, no branching, Z-lines, peripheral multinucleated, voluntary
Actin and myosin are the major contractile proteins
28
Cardiac Muscle
Striations, branching, intercalated disks, mono-nucleated, involuntary
29
What are the 7 types of connective tissue cells?
```
Fibroblasts
Mesenchymal Cells
Adipose
Mast Cells
Macrophages
Plasma Cells
Blood Cells
```
30
Fibroblasts
Main cell type and most common in loose CT
Almost the only cell type in regular CT
Flattened nuclei, spindle shaped cells
31
Mesenchymal Cells
Primitive, relatively undifferentiated
Look very similar to fibroblasts
Differentiate when needed
32
Adipose
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
33
Mast Cells
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
34
Macrophages
"Big eaters"
Scavenger cells that process antigenic material and present it to the lymphocytes
Blood leukocytes are their precursors
35
Plasma Cells
Derived from B lymphocytes
Produce immunoglobulins
Deeply basophilic cytoplasm
36
Blood Cells
Many types
| Red blood cells are the most common
37
What is the basement membrane?
Also called the basal lamina
Between the epithelium and underlying connective tissue
Demonstrated by carbohydrate methods because they contain more sugar than ordinary collagen
38
What is the function of the basement membrane?
Beneath the epithelium and connects it to the underlying connective tissue
Provides physical support to the epithelium, cell attachment, and ultrafiltration
39
What is a unique feature of mast cells that helps in demonstration?
Exhibit metachromasia when stained with Toluidine Blue
40
Which procedures are used to demonstrate lipids?
Oil Red O, Sudan Black, Toluidine Blue
41
Describe Silver Oxidation
Oxidizes reticulin to aldehyde groups
42
Describe Silver Sensitization
Deposit of metallic salts near but not on the reticulin, this metal is later replaced by silver during impregnation
43
Describe Silver Impregnation
Tissue is treated with ammoniacal silver to deposit silver on the reticulin,
44
Describe Silver Reduction
The aldehyde groups in the reticulin reduce the diamine silver to metallic silver
45
Describe Silver Toning
Bound metallic silver is treated with Gold chloride, changing the fibers from brown to black
46
Movat Pentachrome Purpose
Demonstration of mucin, fibrin, elastic fibers, muscle, and collagen
47
Movat Pentachrome Principle
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
Movat Pentachrome Preferred Fixative
10% NBF
49
Movat Pentachrome Basic Procedure
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
Movat Pentachrome Results
```
Nuclei and elastic fibers: Black
Collagen: Yellow
Ground Substance and mucin: blue (or green)
Fibrinoid, fibrin: Intense Red
Muscle: Red (less vibrant)
```
51
Movat Pentachrome Technical Notes
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
Verhoeff Van Gieson Purpose
To demonstrate pathologic changes in elastic fibers; such as atrophy, thinning, loss, breaks, or splitting that may result from vascular diseases
53
Verhoeff Van Gieson Principle
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
Verhoeff Van Gieson Preferred Fixative
Any, but 10% NBF is preferred
55
Verhoeff Van Gieson Basic Procedure
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
Verhoeff Van Gieson Results
Elastic Fibers: Blue-black to black
Nuclei:Blue to black
Collagen: Red
Other tissue elements: Yellow
57
Verhoeff Van Gieson Technical Notes
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
Gomori Reticulin Purpose
Demonstrate reticular fibers, used to diagnose some liver diseases and tumors
59
Gomori Reticulin Principle
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
Gomori Reticulin Preferred Fixative
10% NBF
61
Gomori Reticulin Basic Procedure
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
Gomori Reticulin Results
Reticulin: black
Collagen: taupe
Other tissue elements: pink-red if NFR is used as a counterstain
63
Gomori Reticulin Technical Notes
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
Gordon and Sweets Reticulin Purpose
Demonstrate reticular fibers, used to diagnose some liver diseases and tumors
65
Gordon and Sweets Reticulin Principle
```
Oxidizer: potassium permanganate
Sensitizer: ferric ammonium sulfate
Impregnator: ammoniacal silver
Reducer Formalin
Toner: gold chloride
```
66
Gordon and Sweets Reticulin Preferred Fixative
10% NBF
67
Gordon and Sweets Reticulin Basic Procedure
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
Gordon and Sweets Reticulin Results
Reticulin: black
| Other tissue elements: pink-red if nuclear fast red is used as counterstain
69
Gordon and Sweets Reticulin Technical Notes
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
Mallory PTAH Purpose
Demonstrate muscle cross-striations and fibrin (also glial fibers and myelin); these cross-striations can be found in rhabdomyosarcomas (muscle cell tumors)
71
Mallory PTAH Principle
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
Mallory PTAH Preferred Fixative
Zenker preferred, 10%NBF acceptable
73
Mallory PTAH Basic Procedure
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
Mallory PTAH Results
Cross-striations, fibrin: blue
Nuclei: blue
Collagen: red-brown
75
Mallory PTAH Technical Notes
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
Oil Red O Purpose
Demonstrate neutral lipids in frozen sections
| Can help show fatty emboli due to bone fracture or liposarcomas
77
Oil Red O Principle
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
Oil Red O Preferred Fixative
10%NBF or calcium-formal
| No alcohols, they dissolve lipids
79
Oil Red O Basic Procedure
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
Oil Red O Results
Fat: Red
| Other tissue elements: depends on method used
81
Oil Red O Technical Notes
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
Sudan Black B Purpose
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
Sudan Black B Principle
Soluble in neutral fats, is slightly basic which allows it to combine with acidic groups in compound lipids like phospholipids
84
Sudan Black B Preferred Fixative
10% NBF or post-fixed in calcium-formalin
| NO ALCOHOL
85
Sudan Black B Basic Procedure
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
Sudan Black B Results
Fat: Blue-black
Nuclei: Red
87
Osmium Tetroxide Purpose
Demonstrate fat in a way that allows paraffin embedding
88
Osmium Tetroxide Principle
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
Osmium Tetroxide Preferred Fixative
10%NBF initially, then trim to 2mm thick for osmium tetroxide penetration
90
Osmium Tetroxide Basic Procedure
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
Osmium Tetroxide Results
Fat: black
| Other tissue elements: according to method used
92
Osmium Tetroxide Technical Notes
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
Toluidine Blue Purpose
Demonstrate mast cells in tissue
| Which play a key role in inflammation, allergic reactions, autoimmune disorders, and mastocytomas
94
Toluidine Blue Principle
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
Toluidine Blue Preferred Fixative
10%NBF?
96
Toluidine Blue Basic Procedure
1. Deparaffinize, hydrate
2. Stain in toluidine blue
3. Rinse in distilled water
4. Dehydrate, clear, coverslip
97
Toluidine Blue Results
Mast cells: deep rose-violet
| Background: blue
98
Toluidine Blue Technical Notes
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
Aldehyde Fuschin Stain Purpose
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
Aldehyde Fuschin Stain Principle
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
Aldehyde Fuschin Stain Preferred Fixative
10% NBF, no chromate (B5, Zenker, Helly)
102
Aldehyde Fuschin Stain Basic Procedure
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
Aldehyde Fuschin Stain Results
Elastic Fibers: Deep blue to deep purple
| Other tissue elements: Green
104
Aldehyde Fuschin Stain Technical Notes
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
