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Microtechniques > Connective CH 8 > Flashcards

Connective CH 8 Flashcards

1
Q

Connective and Muscle Tissue Stains

A
  • Connective tissue is ¼ of the basic tissues and functions to provide metabolic support for other tissues and organs
  • Connective tissue neoplasm malignant tumors are termed “scarcomas”
  • Consists of 3 different components: fibers, cells, and amorphous ground substances
  • Connective tissue proper
  • Cartilage
  • Bone
  • Blood
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2
Q

Connective tissue proper: Collagen:

A
  • Collagen: provide strength, very eosiniophilic, found in tendons organ capsules, and the dermis.
  • Is birefringent upon polarization, demonstrated using Masson and Gomori trichrome, and van Gieson stain.
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3
Q

Connective tissue proper: Elastic fibers:

A

◦ Elastic fibers: present in most fibrous connective tissue. Have a flexible property allowing tissue to stretch.

  • Demonstrated by Verhoeff iron hematoxylin, Weigert
    resorcin fuchsin, orecein, or Gomori aldehyde fuchsin stains
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4
Q

Connective tissue proper: Reticular fibers

A
  • Reticular fibers: specific type of collagen fiber. These fibers are ARGYROPHILIC.
  • This is, how the fibers have the ability to ADSORB silver from a solution, but require a chemical reducing agent
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5
Q

Cells of connective tissue proper

A
  • Fibroblasts: most common, produce connective tissue fibers.
  • Mesenchymal cells: develop into various cell types if needed
  • Adipose: fat, store lipid, stain through what?
  • Mast cells: contain histamine and heparin,
    similar to basophils
  • Macrophages: “big eater” cells, not normally
    demonstrated in histopathology lab
  • Plasma cells: derived from b lymphocytes
  • Blood cells: all types
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6
Q

Basement membrane:
Basal lamina

A
  • Basal lamina, seperates epithelium from underlying connective tissue.
  • Provides physical support for epithelium, mainly demonstrated by carbohydrate stains, however silver techniques in this chapter
    can also be used to stain for basement membranes
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7
Q

Muscle tissue
- ¼ of the basic tissue type

A
  • Skeletal muscle: striated, and voluntary. Actin and myosin are major contractile
    components, PTAH can demonstrate cross striations
  • Cardiac muscle: striated, but involuntary. Centrally located nuclei, and contain
    interalated discs
  • Smooth muscle: non striated, and involuntary. Centrally located nuclei, with
    different ratio of actin and myosin.
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8
Q

Connective and Muscle Tissue
Stains
- 14 total

A

1.) Masson trichrome
2.) Gomori 1-step trichrome
3.) Van Gieson
4.) Verhoeff
5.) Gomori Aldehyde Fuchsin
6.) Movat Pentachrome
7.) Gomori silver
8.) Gordon and Sweets silver
9.) Methenamine-silver
10.) Mallory PTAH
11.) Oil Red O
12.) Sudan Black
13.) Osmium Tetroxide
14.) Toluidine Blue

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9
Q

Masson Trichrome Stain
- Demonstrates differentiation between collagen & muscle

A
  • Sections cut at 4 to 5 microns
  • Control tissue: Uterus, small intestine, appendix or fallopian tube
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10
Q

Masson Trichrome Stain

A
  • Reaction is based on three dyes:
    1.) Beibrich scarlet – stains cytoplasm, muscle & collagen

2.) Phosphotungstic acid removes Biebrich from collagen

3.) Aniline blue – stains collagen

4.) Weigert’s Hematoxalyn – stains nuclei

Bouin Fixative used as mordant*

  • Results:
  • Nuclei: Black
  • Cytoplasm, keratin & muscle: Red
  • Collagen & mucus: Blue
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11
Q

Gomori One Step Trichrome
- Demonstrates differentiation between collagen & muscle

A
  • Sections cut at 4 to 5 microns
  • Control tissue: Uterus, small intestine, appendix or fallopian tube
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12
Q

Gomori One-Step Trichrome

A

Uses combined ingredients
- Chromotrope 2R
- Fast-green FCF (or aniline blue)
- Weigerts Hematoxylin

Bouin Fixative used as mordant

Results:
- Nuclei: Black
- Cytoplasm, keratin & muscle: Red
- Collagen and mucus: Green (or Blue)

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13
Q

Verhoeff – van Gieson
- Verhoeff Elastic Stain: Demonstrates elastic fibers

A
  • Van Gieson:
  • Connective tissue stain demonstrating collagen
    & muscle
  • Primarily used as COUNTERSTAIN to Verhoeff
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14
Q

Verhoeff – van Gieson

A
  • Section tissue at 4 to 5 microns
  • Control tissue used:
    1.) Verhoeff – Section of aorta embedded on edge
    (or cross section of large artery).

2.) van Gieson – Uterus, small intestine, appendix
or fallopian tube

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15
Q

Verhoeff Elastic Stain

A
  • Ingredients used:
  • Alcoholic Hematoxylin
  • Ferric Chloride
  • Lugol Iodine
  • Results:
  • Elastic fibers – Blue black to Black
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16
Q

van Gieson
- Ingredients used:

A

Ingredients used:
- Picric acid
- Acid Fuchsin
- Weigert’s Hematoxylin

** Picric Acid must remain acidic to have sharp contrast
between collagen and muscle**

Results:
- Nuclei – Black
- Collagen – Brilliant red
- Muscle & cytoplasm - Yellow

17
Q

Aldehyde Fuchsin Elastic Stain
- Demonstrates elastic fibers

A
  • Demonstrates elastic fibers
  • Avoid chromate fixatives
  • Cut at 4-5um
  • Aorta or skin
18
Q

Aldehyde Fuchsin Elastic Stain
Reagents:

A

◦ Reagents:
- Aldehyde fuchsin solution
- Light green

◦ Results:
- Elastic fiber: deep blue to purple
- Other tissue: green

can also demonstrate ground glass appearance in tissues infected with Hepatitis B
virus

19
Q

Orcein and Reorcein fuchsin

A
  • Orcein , and reorcein fuchsin can also be used. Not common however.
  • Orcein stains elastic fibers brownish
  • Reorcein Fuchsin stains elastic fibers violet to brownish tinge
  • Orcein and Aldehyde Fuchsin methods have been shown to stain Hepatitis B surface antigens
20
Q

Movat Pentachrome
- Demonstrate mucin, muscle fibrin, elastic, and collagen

A
  • Demonstrate mucin, muscle fibrin, elastic, and collagen
  • Cut at 4 – 5 um
  • Control tissue: lung ,skin ,or colon
21
Q

Movat Pentachrome contents

A
  • Alcian blue
  • Alkaline alcohol solution
  • Iodine-Iodine solution
  • Alcoholic hematoxylin
  • Ferric chloride
  • Working hematoxylin
  • Sodium thiosulfate
  • Crocein scarlet acid fuchsin
  • Phosphotungstic acid
  • Alcoholic safran solution
22
Q

Movat Pentachrome
- Results

A

Results:
- Nuclei/elastic fibers – black
- Collagen – yellow
- Ground substance/mucin – blue
- Fibrin – intense red
- Muscle – red

23
Q

Gomori Reticular Stain
- Demonstration of reticular fibers

A
  • Demonstration of reticular fibers
  • Section at 4 to 5 microns
  • Control tissue used: Liver
24
Q

Gomori Reticular Stain
CAN NOT USE METAL FORCEPS!!

  • Silver stain technique used
A
  • Silver stain technique used.
  • Oxidation: produces aldehydes
  • Sensitization: metal impregnation (not silver)
  • Silver impregnation: ammoniacal or diamine silver
  • Reduction: residual silver ions reduced by formaldehyde
  • Toning: gold chloride, turns from brown to black
    3Ag + AuCl3 -> Au + 3AgCl
  • Unreduced silver removed: prevents nonspecific silver
    to stay bound
  • Counterstain: provides contrast
25
Q

Gomori Reticular Stain

A
  • Oxidizer – Potassium permanganate
  • Sensitizer – Ferric ammonium sulfate
  • Impregnation – by aqueous diamine silver solution (ammonical)
  • Reducer – Formalin
  • Toning – Gold chloride
  • Removal – sodium thosulfate
  • Counterstain – Nuclear-Fast Red
  • Results: Reticulin – Black; Collagen - Taupe
26
Q

Gordon and Sweets Reticular stain

A
  • Demonstrated reticular fibers, especially in the liver to aid in the diagnosis of cirrhosis, hepatocellular fibrosis, and necrotic liver disease
  • Control tissues is usually liver
  • Cut at 4 – 5 um
  • Results: reticulin – black, other elements – dependant on counterstain used.
27
Q

Mallory PTAH for cross striations and fibrin

A
  • Demonstrates muscle cross striations used to help diagnose rhabdomyosarcomas. Also
    demonstrates glial fibers and myelin. Replaced today by IHC
  • Control tissue: skeletal or cardiac muscle
  • Cut at 4 – 6 um
  • Results: muscle and fibrin – blue, nuclei – blue,
    collagen – red brown
28
Q

Periodic Acid-Methenamine silver (PAMS)

A
  • Uses METHENAMINE SILVER SOLUTION
  • Demonstrates basement membranes
  • Cut at 2 um
  • Control tissue is kidney.
  • Results: basement membrane – black, background - green
29
Q

Oil Red O for neutral fats
- Demonstrates neutral lipids in frozen tissues

A
  • Demonstrates neutral lipids in frozen tissues
  • No alcohol fixatives should be used. NBF or calcium formol
  • Control, most tissues contain fat, mayonnaise can be used
  • Cut frozen sections at 10um
  • No processing dehydrants can be used, however water soluble waxes CAN be used.
  • Results: fat – red
30
Q

Sudan black
- Demonstrates fat

A
  • Demonstrates fat
  • Most sensitive, can be used to stain phospholipids
  • No alcohol fixatives can be used, NBF or post-fixation in calcium formalin.
  • Staining due to solubility of dye in fat cell
  • Results: fat – blue to black, nuclei – red
31
Q

Osmium tetroxide
- Demonstrates fat in paraffin embedded tissue

A
  • Demonstrates fat in paraffin embedded tissue
  • Chemically combines with fat, not absorbed
  • Tissue is “post-fixed” in osmium tetroxide which actually stains. Routine processing and embedding can be done as normal after
  • Only works on fat droplets, not large amounts of fatty
    tissue
  • Results: fat – black
32
Q

Toluidine Blue for mast cells
- Demonstrates mast cells in tissue

A
  • Demonstrates mast cells in tissue
  • ## Based on the principle of METACHROMATIC STAINING
  • Cut at 4 – 5 um
  • Control tissue should contain mast cells
  • Results: mast cells – deep rose to violet, background - blue

Microtechniques (10 decks)

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