CT, Silver stains, Lipids, Nervous - exam 3

Collagen Type I location:

fibrous supporting tissue, basement membrane, dermis, tendon, ligaments

Collagen Type II location:

hyaline and elastic cartilage

Collagen Type IV location:

basement membrane

Collagen structure and staining:

• Protein and carb content

• Birefringent and acidophilic in H and E

• Stains strongly w/acid dyes

• Very permeable

Elastin location:

Circulatory, integumentary, respiratory systems, bladder, dermis, ligaments, arteries, cartilage, aorta

Elastin structure and staining:

• Branched patterns

• Acidophilic but can become basophilic in solar or actinic elastosis

Solar / actinic elastosis:

Degeneration of elastin fibers in dermis bc of long sun exposure.

Makes elastin basophilic instead of acidophilic

Reticulin location:

Cellular organs as a supportive framework: spleen, LN, liver, kidney, basement membrane, endocrine glands

Reticulin structural and staining:

• Type III collagen

• Argyrophilic

• Weakly birefringent and not ID w/H and E

Fibroblasts

• Most common

• Secrets CT and ECM components

• Is flattened

• Myofibroblasts are active form → scar formation

Mast cells

• Trigger inflammation

• Highly acidic secretory granules w/histamine and heparin

• Exhibit metachromasia

• Are along small blood vessels

• Resemble basophils

Ground substance

Amorphous, non-living substance w/proteoglycans (protein molecules bound to carbs): non-sulfated and sulfated GaGs

Demonstrate w/carb stains

Basement Membrane

Act as growth barrier, support, site of attachment, ultrafiltration in kidney

• Consist of GaGs, Type I, II, IV collagen

• ID w/PAS (carbs) or silver impregnation (reticulin)

• Acidophilic in H&E

Skeletal muscle

voluntary, striated, multi-nucleated

Cardiac muscle

involuntary, striated, branching, multi-nucleated, intercalated discs

Smooth muscle

involuntary, single central nuclei

Masson Trichrome and Gomori 1-Step Trichrome general purpose

differentiate collagen, muscle, fibrin

Masson Trichrome pathology

Assessing fibrosis, scar formation, lesions.

Liver diseases like cirrhosis, hepatitis, etc

Masson Trichrome and Gomori 1-Step Tri. fixatives

Bouins, Zinc formalin

If 10% NBF used, must pretreat with bouins

Masson Trichrome dyes

1. Nuclear stain (optional) - iron hematoxylin

2. Biebrich Scarlet - small acidic dye

   1. Stains all acidophilic structures

   2. Phospho acids differentiates and displaces biebrich from collagen

3. Aniline blue/Light green - large acidic dye

   1. Stains collagen first

   2. Acetic acid rinse removes aniline blue from tissue components OTHER than collagen

Masson Trichrome Results (and Gomori 1-Step results depending on counterstain)

• Collagen and mucin - Blue

• Muscle, epithelium (cytoplasm), fibrin, RBCs - Bright red

• Nuclei if hematoxylin used - blue-black

Why use iron hematoxylin instead of Al hematoxylin?

Al will become decolorized.

Masson Trichrome

Pale nuclear staining (hematoxylin)

• Al hematoxylin used

• Iron hem over oxidized/not fresh

• Low hem time

• Aniline blue not differentiated

Masson Trichrome

Decreased red stain (muscle)

• Expired/overused reagent

• Prolonged water or phospho acid rinse

• Prolonged exposure to dehydrating alcohols

• X pretreatment in Bouin’s (would also lead to bad blue stain)

• Bad fixation

Masson Trichrome

Muscle unstained or gray

• Overstain w/iron hem

• bad reagents

• X treatment with Bouins

Masson Trichrome

Decreased blue staining

• Overdiff in acetic acid (removes blue)

• Pathologically altered collagen (sarcoma) may lose permeability and not bind to aniline blue

Masson Trichrome

Uneven or incorrect staining

• X Bouin’s used

• prolonged fixation

• X differentiation w/phospho acids or acetic acid

• High pH

• Microwave was used

Gomori 1-Step Trichrome solution

• Muscle/plasma = Chromotrope 2R

• Collagen fiber = fast green, light green, or aniline blue are combined with/PTA and acetic acid

Then an acetic acid rinse for differentiation

Gomori 1-Step Trichrome pathology

Distinguishing histological changes in neuromuscular diseases in muscle bxs

Lowering the pH with HCl will ___

Increase the staining intensity of collagen.

Verhoeff van Gieson (VVG), Aldehyde Fuchsin, Orcein, Resorcin-Fuchsin general purpose

Elastic fiber demonstration

VVG pathology

Demonstrating pathological changes in elastic fibers and vascular changes.

Vascular diseases and invasion

Verhoeff elastic stain solution / steps

1. Alcoholic hematoxylin- dye

2. Ferric chloride - oxidizes to hematein and is a mordant

3. Iodine - oxidizes elastic fibers to make them strongly basophilic bc otherwise they are acidophilic

Then differentiation with excess ferric chloride.

Sodium thiosulfate (hypo) - removes stain/iodine

Then van gieson counterstain

Van Gieson staining

• Acid fuchsin - collagen

• Picric acid - muscle, epithelium, RBCs

VVG results

• Elastic fibers - black

• Collagen - red

• Muscle, epithelium, RBCs - yellow

• Nuclei - blue/black

Iodine and hypo will remove what fixation pigment?

Mercury

VVG Pale elastic fibers

• Overdiff w/ferric chloride (can be reversed as long as it hasn’t been in alcohol)

• Verhoeff not fresh/over ox

• Too much time in van Gieson

VVG background staining w/Verhoeff hematoxylin

• Underdifferentiation → fix by placing slides back in ferric chloride

VVG Poor counterstain

• Rinsed after van Gieson (not supposed to do)

• Expired/overused van G.

• Over/under stain

Aldehyde Fuchsin results

Elastin = purple

Background = green or red-yellow

Orcein results

Elasin = red/brown

Background = counterstain

• Also will stain copper-associated protein and hep B surface antigen

Resorcin-Fuchsin results

Uses resorcin and basic fuchsin w/ferric chloride

Elastin = blue-black

Collagen = red

Smooth muscle = yellow

• Looks like VVG

Movat Pentachrome general purpose

Differentiate mucin, fibrin, elastin, muscle, collagen

Plus fungi bc of AB

Movat Pentachrome pathology

Fibrosis and elastic fiber changes in vasculature in temporal arteries

Movat Pentachrome AB steps/reagents

1. AB dye stains acidic glycoproteins and GaGs

2. Alkaline alcohol makes AB an insoluble monastral fast blue

3. Remove alkaline alcohol or it inhibits staining

Movat Pentachrome Elastic fibers steps

4. Iron hematoxylin (alcoholic hematoxylin and ferric chloride) stains nuclei and elastic fibers

5. Excess mordant to differentiate

6. Sodium thiosulfate (Hypo) rinse to differentiate excess mordant/stain

Movat Pentachrome Collagen fibers steps

7. Biebrich, Crocein, or Woodstain scarlet acid dye stains muscle and collagen

8. PTA displaces acid from collagen adn ground substance

9. Acetic acid rinse removes PTA

10. Alcohol rinse prevents carryover of acetic acid into safran/saffron

11. Alcoholic safran/saffrom acid dye stains collagen

Movat Pentachrome results

• Nuclei and elastic fibers = black

• Collagen = yellow

• Mucin, GaGs, fungus = blue

• Fibrin = intense red

• Muscle = red

Safran/saffron notes

• Safran/saffron can be re used

• Can be microwaved

• Must be anhydrous

• Dehydrate with only 100% alcohol bc aq alcohols will remove safran/saffron dye

Argyrophilia

tissue binds with metal but cannot reduce them to their metallic visible form

Argentaffin

tissue binds to metal but can reduce it to the metallic visible form

Silver techniques advantages

• Stable, permanent end product that doesn’t fade

• Black deposit is excellent contrast

• Silver will detect minute amounts of fibers

Silver techniques disadvantages

• Inconsistant

• Tissue fragmentation

• Susceptibility to nonspecific or background staining

• Hard to remove from equipment, clothing, skin…

• Sensitive to contamination

• Can be explosive

• Expensive

• Cannot use metallic instruments or supplies

Oxidation in silver techniques

• Oxidation - reticulin fibers are oxidized to aldehydes

  • Phosphomolybdic acid

  • K permanganate (brown color removed w/K metabisulfite or oxalic acid)

  • Periodic acid/chromic acid

Sensitization/metallic impregnation in silver techniques

Deposition of metallic salts (silver or ferric) on/around of aldehyde groups

• Need high pH

• Enhanced sensitivity and selectivity

• Ferric ammonium sulfate (iron alum), uranyl nitrate (uranium metal ion), dilute silver nitrate

Silver impregnation in silver techniques

Silver ions replace metallic salts in addition to reacting with any remaining exposed aldehydes.

• Best at high pH

• Silver solutions: ammonical silver, silver nitrate, methenamine silver

Reduction/Developing in silver techniques

Silver ions are reduced to a visible brown precipitate.

• Unbuffered formalin (reticulin fibers and nerve)

• Hydroquinone (microorganisms and nerve)

• Light-artificial or natural (used for Ca demonstration)

Toning in silver techniques

Silver ions are replaced with gold ions forming a more intense, stable black precipitate.

• Toner: Gold chloride

• Overtoning- individual deposited gold ions will appear purple. Aggregation = black

  — individual gold ions being deposited nonspecifically giving the background a purple color

  — Excess gold removed with 3% Na metabisulfate

Na thiosulfate (hypo) ___

dissolves unreduced silver ions preventing nonspecific staining.

Counterstains in silver techniques

Optional

• NFR, eosin, tartrazine, van Gieson, light green

Gormori Reticulin and Gordon and Sweets general purpose

Show reticular fibers

Gormori Reticulin pathology

BM fibrosis, myeloproliferative disorders, Acute leukemia

Gormori Reticulin steps

1. Oxidizer - K permanganate

2. Bleaching - K metabisulfite

3. Sensitizer - ferric ammonium sulfate

4. Silver impregnation

   • Ammoniacal/diamine silver solution.

   • Add ammonium hydroxide until precipitate clears

   • Add 1-2 drops of silver nitrate until cloudy to absorb excess ammonia.

5. Reducer - unbuffered formalin

6. Toner - gold chloride

7. Unreduced silver rinse - hypo

Gormori Reticulin QC

liver, spleen, tonsil, LN

Gormori Reticulin results

• Reticulin fibers - black

• Nuclei/background - gray

Lipofuscin and silver

They will rx so some cytoplasmic granularity is expected, especially in liver

Ammonical/diamine silver solution in a Gomori reticulin stain

K hydroxide + silver nitrate → silver oxide precipitate

Then add ammonium hydroxide until precipitate clears

Gordon and Sweets

Same as Gomori except oxalic acid is a bleaching solution instead of potassium metabisulfate

Periodic acid-methenamine silver method (PA-MS)/Jones basement membrane stain purpose

show basement membranes

PA-MS/Jones pathology

glomerulosclerosis/nephritis, renal disease

PA-MS/Jones principle/steps

Ox - Periodic acid

NO SENSITIZER

Silver impregnation - heated methamine silver

• Silver nitrate + aq methenamine and borax as buffers

NO REDUCER

Toner - gold chloride

Hypo rinse

Counterstain w/light green (most common), NFR, H&E (Jones)

Note: less alkaline than ammonical silver solutions and temp of silver should be 95 degrees C.

Heated methamine silver…

Heat makes methamine into ammonia and formaldehyde.

Formaldehyde → red silver to precipitate

PA-MS results

Basement membrane - black

Background - green

Phosphotungstic acid hematoxylin (PTAH) stain purpose

Cross-striations and fibrin PTAH

PTAH pathology

rhabdomyosarcomas, microthrombi

PTAH principle/reagents

Tungsten mordanted hematoxylin: hematein + phosphotungstic acid will stain cross-striations, fibrin, and nuclei.PTAH

PTAH best fixative

Zenker but NBF ok.

If formalin fixed, mordant in bouins or zenker. PTAH

PTAH results

• Cross striations - blue-black

• Collagen - red-brown

MSB (Lendrum) purpose

Fibrin stages

MSB (Lendrum) pathology

distinguish early, intermediate, old deposits

MSB (Lendrum) prinicple/reagents

Maritus yellow → small dye → Early deposit

Scarlet → intermediate size dye → middle-aged deposits

Methyl Blue → large dye → collagen plus old deposits

MSB (Lendrum) results

• Early fibrin and RBCs - Yellow

• Middle aged and muscle - red

• Older and collagen - blue

Toluidine Blue purpose

mast cells (histamines and haparin), GaGs in mast cell granules, cartilage matrix

Toluidine Blue pathology

inflammatory conditions, mastocytomas

Toluidine Blue reagents

Metachromatic dye will demonstrate mast cell granules.

Note that methylene blue and pyronin Y will also demonstrate mast cells.

Toluidine Blue results

• Mast cell granules - deep rose-violet (metachromasia)

• Background - blue (orthochromasia)

All lipids are…

soluble in organic solvents and insoluble in water.

Simple/Neutral lipids are found…

in adipocytes and lipid vacuoles in cytoplasm

Compound lipids are…

phospholipids (cell membranes), lipofuscin, and myelin.

Derived lipids are:

Cholesterol, bile acids, hormones

Oil Red O purpose

simple lipids

Oil Red O pathology

lipid storage diseases, liposarcomas

Oil Red O principles

Hydrophobic interaction btwn oil soluble dye and lipids.

Dye characteristics-

• More soluble in lipids that solvent

• NOT water soluble

• Strongly covered

Solvents for lipid dye: isopropanol or propylene glycol

Isopropanol as a solvent

Removes minimum amount of lipids from tissue

Propylene glycol as a solvent

does not remove any lipids (preferred)

Oil Red O fixative and technique

Just NO alcoholic fixatives

Frozens (can improve w/30% sucrose) or tissues in water soluble wax (carbowax)

QC = fat

Oil Red O results

• Lipids = red

• Background = blue-purple

Other info about Oil Red O

• Mounting media: aqueous only bc synthetic will dissolve lipids. Bc aq used, must seal

• No dehydrating alcohols or xylene

• No pressure when coverslipping

Sudan Black B and Osmium tetroxide purpose

simple AND compound

Sudan Black B and Os tetroxide pathology

lipids storage diseases, liposarcomas, degenerative changes in myelin, leukemias.