Chapter 7: Carbohydrates and Amyloid Flashcards
What is a carbohydrate?
hydrated carbons which are organic compounds such as sugars, starch, cellulose, and polymers linked to protein
What are the 3 types of Carbohydrates?
Monosaccharides: one sugar unit
Oligosaccharides: few, 2-10 sugar units
Polysaccharides: many sugar units
What is glycogen?
the polymer form of glucose used to store carbohydrates/energy
2 places where glycogen is located
Liver and skeletal muscles
Why is fixation important when staining for glycogen?
Because glycogen is quickly broken down to glucose after death
What is a polysaccharide?
a carbohydrate containing many sugar units, such as glycogen
What are acid mucosubstances?
Refers to Group 2 acid mucopolysaccharides and Group 3 glycoproteins
Both stain with alcian blue, but not all stain with PAS
epithelial or connective tissue mucins
What are the 4 groups of natural polysaccharides?
Group 1: Neutral Polysaccharides (nonionic homoglycans)
Group 2: acid mucopolysaccharides (anionic heteroglycans)
Group 3: Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances)
Group 4: Glycolipids
What is glucose and its characteristics
free floating monosaccharide abundant in the body
Because glucose is soluble in aqueous solutions and small it cannot be demonstrated in tissue sections
What is mucin, and what are its properties?
Large, heavily glycosylated proteins secreted by epithelial and connective tissues in most humans
PAS positive
most are metachromatic and basophilic
precipitated by acetic acid
soluble in alkaline solutions (only use tap water for bluing)
Birefringence
Light is split into 2 waves refracted in different directions
Especially relevant for visualizing amyloid deposits in the Congo red stain under polarized light
Metachromasia
Change of color
Some tissue elements will stain a different color from the dye, and the background will stain the expected color
Ex: Toluidine blue on mast cell granules stains red to purple against a blue background
Polychromasia
A single solution that stains various tissue elements different colors based on which elements of the dye they interact with. This is not metachromasia
What is amyloid?
“Starchlike”
A fibrillar protein that deposits in tissue under certain pathogenic conditions and contains 1-2% carbohydrate
Where is amyloid typically found in the body?
Extracellular space of organs
What is amyloidosis?
Deposition of amyloid in extracellular space that gradually replaces cellular elements of organs eventually leading to death
What are the four old groups of amyloid?
Primary: spontaneous without prior disease; muscle, heart, skin, tongue
Secondary: usually associated with inflammatory disease; kidney, liver, spleen, and adrenal glands
Myeloma: associated with diseases of the immune system; muscle, heart, skin, tongue
Tumor associated: esp with tumors of the amine precursor uptake and decarboxylation system
What is the current number of amyloid types?
20
How are amyloids currently classified?
Abbreviation of originating protein, preceded by an A
How to test the quality of Schiff Reagent?
10mL of 37-40% formaldehyde in a beaker
Add a few drops of Schiff’s reagent
rapid reddish purple is good
slow bluish purple is bad
PAS detects which type of carbohydrate?
glycogen
polysaccharides such as glycogen, mucosubstances such as glycoproteins, glycolipids, and mucins in tissues
PAS requires which fixative?
10% NBF (or Bouin’s ONLY IF NOT DIGESTING)
PAS Purpose
demonstrate polysaccharides, neutral mucosubstances, and basement membranes
PAS Principle
Periodic acid oxidizes reactive groups to form aldehydes (-CHO)
Basic fuschin + sulfurous acid = leucofuschin/Schiff’s reagent which binds to exposed aldehyde groups
Water washes away the sulfur resulting in the rose chromophore
PAS Basic Procedure
- Deparaffinize and hydrate
- 0.5% Periodic acid for 5 minutes
- Wash with distilled water 3X
- Stain with Schiff reagent for 15 minutes
- Wash in 0.55% potassium metabisulphate to remove nonspecific Schiff background staining
- Wash in running tap water for 10 min to develop full color
- Counterstain 30 seconds in Harris Hematoxylin with acetic acid
- Wash sections well to blue the Hematoxylin
- Dehydrate, clear, coverslip
PAS Results
Glycogen, neutral mucosubstances, some epithelial sulfomucins and sialomucins, thyroid colloid, basement membranes, and fungal walls stain Bright Rose
PAS Technical Notes
Make sure to test Schiff: fast red purple is good, slow blue purple is bad
Fast green is an alternative counterstain to Hematoxylin
Don’t fix with glutaraldehyde because it causes non-specific binding of Schiff’s
Liver with abundant glycogen is a bad positive control because of abundance makes stain visualization difficult
PAS with Diastase digestion detects which type of carbohydrate?
Specifically glycogen, more specific than PAS alone
PAS-D requires which fixative?
10% NBF, formalin alcohol, or 100% alcohol
PAS-D Purpose
demonstrate glycogen in tissue sections (by comparing to PAS without digestion)
PAS-D Principle
This is a very sensitive reaction
Diastase and a-amylase depolymerize glycogen into glucose and maltose subunits that wash out of the section during the reaction, while mucin in glands remains polymerized
Schiff reaction is the same as in PAS
PAS-D Basic Procedure
- Deparaffinize, hydrate
- digest slide in diastase or saliva (a-amylase) for 20 minutes at room temp
- Wash in running water for 5 minutes
- 0.5% periodic acid for 5 minutes
- Wash in distilled water 3X
- Stain with Schiff for 15 minutes
- Wash for 1 minute in potassium metabisulfate to reduce background and non-specific staining
- Wash in running water for 10 minutes
- Counterstain for 30 seconds in Harris Hematoxylin
- Wash in running water
- Dehydrate, clear, coverslip
PAS-D Results
Positive rose stain for Glycogen is pale compared to undigested PAS stained slides which are Bright Rose
PAS-D Technical Notes
A-amylase is preferred because it doesn’t lift tissue off slides like diastase
Picric acid fixative (ex Bouin’s) can cause glycogen to be resistant to digestion and give a false result
Best Carmine detects which type of carbohydrate?
Glycogen (Group 1)
Best Carmine requires which fixative?
Absolute alcohol (Carnoy and Bouin’s are also acceptable)
Best Carmine Purpose
Demonstrate glycogen (inferior to PAS)
Best Carmine Principle
High pH of the staining solution (9-11) allows the dye to hydrogen bond to glycogen
Best Carmine Basic Procedure
- deparaffinize, hydrate
- Stain with Mayer or Harris Hematoxylin
- Wash in running water
- Stain in carmine solution (contains ammonium hydroxide, hazardous, use in hood)
- Differentiating solution
- 80% alcohol rinse
- Dehydrate, clear, coverslip
Best Carmine Results
Glycogen: Pink to red
Nuclei: Blue
Best Carmine Technical Notes
PAS is better because its more specific for glycogen, and the ammonia solutions in Best carmine are hazardous, and evaporate easily
Important to obtain fresh liver for controls because glycogen quickly breaks down after death
Mayer Mucicarmine detects which type of carbohydrate?
“epithelial” mucins and Cryptococcus neoformans fungus (Group 3)
Mayer Mucicarmine requires which fixative?
10% NBF
Mayer Mucicarmine Purpose
Stain epithelial mucins, esp goblet cells
carboxylated and sulfonated (acidics) nut not neutrals
Mayer Mucicarmine Principle
Stains carboxylated and sulfonated mucins by attaching to the acid groups of mucins through an aluminum chelation complex, thus does not stain neutral mucins
Mayer Mucicarmine Basic Procedure
- Deparaffinize, hydrate
- Stain in Weigert (Iron) Hematoxylin (resists destaining in subsequent acidic solutions)
- Wash in running water (helps with bluing)
- Stain in mucicarmine solution
- Rinse and remove excess water
- Counterstain in metanil yellow (careful not to overstain)
- Dehydrate, clear, coverslip
Mayer Mucicarmine Results
Mucin: Deep rose to red
Cryptococcus capsule: deep rose to red
Nuclei: black/grey
Other tissue elements: blue (Gill Hematoxylin) or yellow
Mayer Mucicarmine Technical Notes
Only rinse in tap water because mucins are soluble in alkaline solutions
don’t overstain with metanil yellow or you may mask the mucicarmine
mucicarmine stock deteriorates over time, monitor your control slides for gradual fading as your stock ages
prepare stock under hood because it can release HCl vapors
Alcian Blue pH 2.5 detects which type of carbohydrate?
Group 2 Acid mucoploysaccharides, also known as connective tissue mucins
carboxylated only
carboxylated and sulfonated
sulfonated only
Alcian Blue pH 2.5 requires which fixative?
10% NBF or Bouin’s
Alcian Blue pH 2.5 Purpose
demonstrate acid mucopolysaccharides
Alcian Blue pH 2.5 Principle
Alcian blue is a water soluble copper dye
In a 3% acetic solution Alcian blue stains both sulfated and/or carboxylated acid mucopolysaccharides and sulfated and/or carboxylated glycoproteins
Alcian blue forms salt linkages with the acidic groups of acid mucopolysaccharides
Alcian Blue pH 2.5 Basic Procedure
- Deparaffinize, hydrate
- 3% acetic acid (condition the tissue)
- Alcian blue
- 3% Acetic acid rinse (prevents nonspecific staining)
- Wash in running tap water, warm preferred
- Rinse in distilled water
- Counterstain in Nuclear Fast Red
- Wash in running tap water to prevent cloudiness caused by NFR mixing with alcohol
- Dehydrate, clear, coverslip
Alcian Blue pH 2.5 Results
Weakly acidic sulfated mucosubstances: dark blue
Hyaluronic acid: dark blue
Sialomucins: dark blue
Background: pink to red (may have some light blue areas)
Nuclei: Red
Alcian Blue pH 2.5 Technical Notes
3% Acetic acid before Alcian blue stain conditions tissue to be more dye receptive
Incomplete dehydration prior to staining leads to weak staining
Overstaining with Alcian blue leads to blue nuclei (which should be red)
Sections that are not washed well after Nuclear Fast Red counterstain will become cloudy because it reacts with the alcohol dehydrant prior to coverslipping
Alcian Blue pH 1.0 detects which type of carbohydrate?
Sulfated mucosubstances including:
Group 2 acid mucopolysaccharides and Group 3 glycoproteins
Alcian Blue pH 1.0 requires which fixative?
10% NBF or Bouin’s
Alcian Blue pH 1.0 Purpose
Demonstrate sulfated mucosubstances, rarely used, image is crisper than pH2.5
Alcian Blue pH 1.0 Principle
At pH1 Alcian blue only stain sulfated acid mucopolysaccharides and sulfated glycoproteins, not unsulfated ones
carboxylated groups aren’t stained because they can’t ionize at pH1
Alcian Blue pH 1.0 Basic Procedure
- Deparaffinize, hydrate
- Rinse in 0.1N HCl (instead of Acetic acid) conditioning
- Stain in 1% Alcian blue in 0.1N HCl for 30 minutes
- Rinse in 0.1N HCl (reduces nonspecific staining)
- Blot sections dry (reduces nonspecific staining)
- 5 minute counterstain in Nuclear Fast Red
- Wash in distilled water (to prevent cloudy reaction)
- Dehydrate, clear, coverslip
Alcian Blue pH 1.0 Results
Sulfated mucosubstances: pale blue
Background: pink to red
Nuclei: red
Alcian Blue pH 1.0 Technical Notes
Same as pH2.5
Section is blotted dry after HCl rinse because washing it in water could cause a pH change and nonspecific staining
Alcian Blue with Hyaluronidase digestion detects which type of carbohydrate?
Group 4 Glycoproteins, also known as epithelial mucins
Alcian Blue with Hyaluronidase digestion requires which fixative?
10% NBF
Alcian Blue with Hyaluronidase digestion Purpose
Differentiates epithelial mucins from connective tissue mucins
Alcian Blue with Hyaluronidase digestion Principle
Alcian blue reaction is the same as previous protocols
Blue staining is dramatically reduced for connective tissue mucins such as: hyaluronic acid, chondroitin sulfate A, and chondroitin sulfate C
While stain remains strong for epithelial mucins such as Group 4 glycoproteins
Alcian Blue with Hyaluronidase digestion Basic Procedure
- Deparaffinize, hydrate
- Digest slides in hyaluronidase 37C for 2 hours
- 5 minute wash in running water
- 3% acetic acid (condition the tissue)
- Alcian blue
- 3% Acetic acid rinse (prevents nonspecific staining)
- Wash in running tap water, warm preferred
- Rinse in distilled water
- Counterstain in Nuclear Fast Red
- Wash in running tap water to prevent cloudiness caused by NFR mixing with alcohol
- Dehydrate, clear, coverslip
Alcian Blue with Hyaluronidase digestion Results
Without digestion: deep blue acid mucopolysaccharides and sialomucins
with digestion: pale blue mucosubstances that contain hyaluronic acid and chondroitin sulfates A and C
Nuclei: reddish pink
Alcian Blue with Hyaluronidase digestion Technical Notes
3% Acetic acid before Alcian blue stain conditions tissue to be more dye receptive
Incomplete dehydration prior to staining leads to weak staining
Overstaining with Alcian blue leads to blue nuclei (which should be red)
Sections that are not washed well after Nuclear Fast Red counterstain will become cloudy because it reacts with the alcohol dehydrant prior to coverslipping
Alcian Blue-PAS-Hematoxylin detects which type of carbohydrate?
Acidic and neutral mucosubstances
sometimes abbreviated PAB or AB-PAS
Alcian Blue-PAS-Hematoxylin requires which fixative?
10% NBF or Zenker
Alcian Blue-PAS-Hematoxylin Purpose
Differentiates between neutral and acidic mucosubstances
Used to detect intestinal metaplasia
Alcian Blue-PAS-Hematoxylin Principle
Alcian blue stains the acidic mucosubstances
PAS stains the neutral mucosubstances
Alcian Blue-PAS-Hematoxylin Basic Procedure
- Deparaffinize, hydrate
- 3% acetic acid (conditioning)
- Stain with alcian blue
- Wash in tap water, rinse in distilled water
- Incubate in periodic acid (to form aldehyde groups)
- Wash in tap water, rinse in distilled water
- Stain with Schiff reagent
- Reducing rinse of sodium metabisulfate (to prevent nonspecific PAS staining)
- Wash in running tap water
- Stain with Harris Hematoxylin
- Dehydrate, clear, coverslip
Alcian Blue-PAS-Hematoxylin Results
Exclusively acid mucosubstances: blue
Neutral polysaccharides: magenta
some substances will stain purple because they react to both PAS and alcian blue
Alcian Blue-PAS-Hematoxylin Technical Notes
Same as PAS and Alcian blue pH2.5
Colloidal Iron detects which type of carbohydrate?
carboxylated and sulfated mucopolysaccharides (group 2) and glycoproteins (Group 3)
Colloidal Iron requires which fixative?
10% NBF (Avoid chromate fixatives B-5, Zenker, Helly)
Colloidal Iron Purpose
Demonstrate carboxylated and sulfated mucopolysaccharides (group 2) and glycoproteins (Group 3)
Colloidal Iron Principle
At low pH carboxylated and sulfated mucosubstances absorb Colloidal iron ions. Then Prussian blue reaction detects those bound iron ions to indirectly demonstrate the presence of acid mucosubstances
Colloidal Iron Basic Procedure
- Deparaffinize, hydrate
- 12% acetic acid rinse (prevents water dilution of colloidal iron solution)
- Stain (primary) in colloidal iron
- 12% acetic acid rinse (for specificity)
- Ferrocyanide-HCl solution (secondary) (also used in Gomorri’s Iron stain)
- wash in running tap water (for specificity)
- Counterstain in Nuclear Fast Red
- Wash in running water (to prevent cloudiness)
- Dehydrate, clear, coverslip
Colloidal Iron Results
Acid mucopolysaccharides and Glycoproteins (sialomucins): deep Prussian Blue
nuclei: pink-red
Cytoplasm: pink
Cryptococcus neoformans fungus: deep blue
Colloidal Iron Technical Notes
A control should be run without the initial colloidal iron solution to ensure that Prussian Blue staining is not sue to hemosiderin (excess iron deposits in the tissue)
Less specific than alcian blue for acid mucopolysaccharides
May have some background due to connective tissue mucins
Strongly acid mucins that do not stain with Alcian also won’t stain with colloidal iron
Crystal Violet detects which type of carbohydrate?
Amyloid
Crystal Violet requires which fixative?
10% NBF or alcohol
Crystal Violet Purpose
Faster but less accurate way to detect amyloid, good for rapid screening
Crystal Violet Principle
Polychromatic stain, amyloid stains rose due to reacting with one of the components of crystal violet
Crystal Violet Basic Procedure
- Deparaffinize, hydrate
- Stain in working crystal violet
- Rinse in tap water
- Mount in Apathy’s or air dry and mount with synthetic resin
Crystal Violet Results
Amyloid: purplish violet
Other tissue elements: Blue
Crystal Violet Technical Notes
Bleeding into the mounting media can be prevented by adding sugar, this is why Apathy’s is preferred for aqueous mounting
staining in crystal violet for 5 hours can increase intensity and sensitivity of the stain because it self differentiates
some procedures use a 1% acetic acid wash to differentiate
Congo Red detects which type of carbohydrate?
Amyloid
Congo Red requires which fixative?
Alcohol or Carnoy preferred (10% NBF acceptable)
Congo Red Purpose
Best stain to demonstrate amyloid
Congo Red Principle
Benzidine derivative that reacts with cellulose (amyloid is similar to cellulose). Alkali acids separate protein chains exposing hydroxyls on the amyloid, which hydrogen bond to azo and amide groups in the Congo Red dye. The dye binds with the polysaccharide or protein component of amyloid. Results in highly specific apple green birefringence.
Congo Red Basic Procedure
- Deparaffinize, hydrate
- Stain in Harris Hematoxylin with acetic acid
- Wash in running tap water
- Alkaline salt solution (makes hydroxyls of amyloid available)
- Stain in working Congo Red solution
- Dehydrate, clear, coverslip
Congo Red Results
Amyloid: deep pink to red, apple green birefringence under polarized light
Elastic tissue: pink
Nuclei: blue
Congo Red Technical Notes
must cut sections 8-10uM for birefringence, too thin results in red, too thick results in yellow
Thioflavine T Fluorescent Method detects which type of carbohydrate?
Amyloid
Thioflavine T requires which fixative?
10% NBF
Thioflavine T Purpose
Demonstrate amyloid, not as specific as Congo Red
Thioflavine T Principle
Fluorescent dye that attaches to amyloid. Background nuclear fluorescence is quenched by staining with aluminum Hematoxylin
Thioflavine T Basic Procedure
- Deparaffinize, hydrate
- Stain in Mayer hematoxylin to quench nuclear fluorescence
- Wash in water
4 Stain in filtered Thioflavin T - Rinse in distilled water
- Differentiate in 1% acetic acid
- Wash in running water and blot dry
- Mount with a non-fluorescent mountant
Thioflavine T Results
Amyloid: fluoresces yellow to yellow green
Group 1: Neutral Polysaccharides (nonionic homoglycans)
Glucose containing: glycogen, starch, cellulose
N-acetyl-glucosamine-containing: chitin
Very positive PAS stain
Negative for alcian blue, colloidal iron, and mucicarmine
Group 2: acid mucopolysaccharides (anionic heteroglycans)
They are all acidic (anionic) polysaccharides attached to protein
Connective tissue mucins
PAS stains negative
Stains positive for alcian blue, colloidal iron, and mucicarmine
Group 3: Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances)
Mostly epithelial mucins, but some may occur in connective tissue
Potentially PAS positive, but not always
Group 4: Glycolipids
Cerebrocides: fatty residue on a carbohydrate structure
phosphatides: PAS positive lipids that don’t contain carbohydrates
PAS positive, may react to other stains
PAS (incorrectly) stains acid mucopolysaccharides
skipped the metabisulfate wash and then rinsed in over-chlorinated tap water causing non-specific off-target staining
PAS/D (incorrectly) shows glycogen staining
glycogen wasn’t properly digested by diastase
Probably due to fixation in bouins/picric acid which inhibits diastase
Background on mucicarmine is nearly completely yellow
overstained with metanil yellow
Alcian blue shows non-specific staining
the stain is underdifferentiated, pretreat and rinse longer in acetic acid if staining with pH2.5, 1NHCL if using pH1.0
Amyloid shows faint red birefringence
sections are too thin, make sure they are 8-10uM for Congo Red stain
Congo red shows unnecessary background staining
make sure that the alkaline salt solution component is properly and fully saturated to prevent retention of excess Congo red dye in the tissue
PAS control slide for fungi is very pale
make sure to counter stain with fast green
make sure not to use chromate fixatives (like B5, Zenker, or Helly) which may overoxidize reactive groups and lead to a weakened schiff reaction
A section mounted with synthetic resin appears cloudy
If nuclear Fast Red counterstain was used, the slide probably wasn’t properly rinsed in water before dehydrating
remove the coverslip, back up to the NFR step, then make sure to thoroughly rinse in running tap water before dehydrating and coverslipping
Excessive hydrolysis seen in a Feulgen Reaction
Probably used a picric acid fixative (like bouins) which overhydrolyzed the DNA resulting in a poor Feulgen stain
PAS -
Alcian pH2.5 +
Colloidal Iron +
not neutral, not glycogen, not sialomucin
acid mucopolysaccharide “connective tissue mucin” carboxylated or sulfonated, or glycoprotein (sialomucin) “epithelial mucin”
acid mucopolysaccharide “connective tissue mucin”, carboxylated or sulfonated, or glycoprotein (sialomucin) “epithelial mucin”
These results suggest the presence of an acid mucosubstance (Probably Group 2), carboxylated or carboxylated and sulfated since pH1.0 wasn’t performed to demonstrate whether it is sulfonated only
