Carbohydrates Staining Flashcards
What (PAS, AB, Mucicarmine) is pos or neg for staining neutral polysaccharides (neutral mucins)?
PAS - POS
Alcian Blue - NEG
Mucicarmine - NEG
What is pos or neg for staining Acid mucopolysaccharides (aka acid mucins)?
PAS - NEG
Alcian Blue - POS
Mucicarmine - POS
What techniques stain glycoproteins?
PAS - POS (but not always)
Alcian Blue - NEG
Mucicarmine - NEG
How well do glycolipids stain with PAS, AB and Mucicarmine?
Glycolipids stain variable with the PAS, AB and Macicarmine techniques.
Phosphatides are PAS positive in non carbohydrate containing lipids.
What will Periodic Acid Schiff’s technique (histochemical) stain?
Periodic Acid Schiff’s will demonstrate:
- Glycogens and Neutral mucins.
- Mucoproteins and glycoproteins.
- Glycolipids.
What are the order of reagents and their purpose in Periodic Acid Schiff’s histochemical technique?
- Periodic acid - 1st Oxidizer
- Schiff’s reagent
- Running Tap Water - 2nd Oxidizer
- Harris Hematoxylin - Counterstain
What is the carbohydrate demonstration with Schiff’s reagent dependent on?
Depends on the conversion of glycols of the various polymers of glycogen and neutral mucopolysaccharides to aldehyde and their subsequent reaction with Schiff’s reagent to form a permanent, highly coloured, insoluble reaction product.
What chemical components make up Schiff’s reagent?
- Schiff’s reagent is prepared by treating basic fuchsin with sulfurous acid.
- When acid is added the chromophore in the basic fuchsin (red solution) is removed and the solution becomes colorless (clear).
What is included in the Harris Hematoxylin solution to reduce the number of steps in the technique but to ensure proper staining?
In PAS, when Harris Hematoxylin is used as a counterstain we used Glacial Acetic Acid which eliminated the need to use both Acid Alcohol and Ammonia water in the technique.
Why does the procedure require the use of distilled water for PAS?
Histochemical reactions require distilled water so there isn’t any metal ions substances that will cause non-specific staining on the tissue.
Also recommended to use plastic forceps instead of metal.
What type of glassware is recommended for PAS and why?
Acid washed glassware.
Similarly to reason for using distilled water, it is to ensure no nonspecific staining occurs on the tissue due to contaminants.
Why should you be really careful to avoid contact with Schiff’s Reagent on your hands and clothing?
It is initially colourless, but when it makes contact with water (like during washing) it will turn bright pink (same as it does in the procedure!).
It is important to use fresh Schiff’s reagent, how can you test to make sure your reagent is fresh?
Potency Test-
1. Place 10 ml of 37%-40% formaldehyde in a small beaker or Erlenmeyer flask.
2. Add a few drops of Schiff’s to Formaldehyde.
3. Interpret change in solution colour as follows:
Reddish-purple –> good to use.
Deep blue-purple (slowly) –> Do NOT use.
What is the purpose of creating a diastase control slide?
A diastase control slide helps determine if your non-control slide is staining glycogen or not. The control slide is put in a solution with amylase enzyme to break down glycogen and removes all glycogen.
Result is a slide that shows hematoxylin staining nuclei and background of tissue with NO MAGENTA PINK.
What is streaming?
Streaming occurs when the tissue is fixed with 10% Formalin.
To make room on the cells for the Formalin to fix tissue properly the glycogen in each cell is pushed to the side of the cell and proper fixation can occur.
Not a bad thing, it just happens, recognize it when it happens.
What does Alcian blue stain?
Stains acid mucins
What does different pH in the alcian blue technique differentiate?
pH will differentiate types of acid mucins:
- pH 2.5 OSO3H & COOH stain sulphated and carboxylated acid mucins
- pH 1.0 OSO3H ONLY stains only sulphated acid mucins
What else does Alcian Blue stain besides acid mucins?
Mast cells, cartilage, goblet cells, etc.
What are the main steps with Alcian blue technque?
Main Steps
- Pre rinse with acid solution –> H+ neutralize other acid groups
- Alcian Blue –> Stain Acid Mucins (& mast cells, cartilage) turquoise blue.
- Harris Hematoxylin – Counterstain (stains nuclei blue), next two steps are same as for H&E as done after hematoxylin (except no Eosin).
a) 0.5% Acid alcohol - differentiator (selectively removes background colour)
b) Ammonia water (bluing agent - intensifies stain of nuclei)
Why does Alcian Blue dye not stain nuclei?
Dye molecule too big to penetrate nuclei. Counterstain of Harris Hematoxylin used to stain nuclei.
What are some other counterstains that could be used instead of Harris Hematoxylin?
Other counterstain options – Neutral Red (G&S), Nuclear Fast Red
What is the chemical make-up of Alcian Blue and its characteristics?
Alcian Blue:
- Copper phthalocyanine, basic dye.
- Water soluble.
- Coloured blue because of copper content.
Note: Similar to Luxol Fast Blue for myelin.
Why is an acid rinse before and after using alcian blue recommended?
- Rinsing slides with acid before Alcian Blue solution will protect a solution that is used repeatedly from pH changes because of the introduction of water.
- Rinsing slides with acid after Alcian Blue will prevent non-specific staining.
Conclusion –> Improves results.
Why is hydrating slides really important for the alcian blue technique?
During take down hydrating the slides well is important for Alcian Blue staining, because some ALCIANOPHILIC structures HYDRATE SLOW. If not hydrated properly, result would be WEAK STAINING.
Why should slides not be left too long in Alcian blue before doing nuclear staining?
Slides left in Alcian Blue for a very long time can cause nuclear staining.
Why would Alcian Blue and Periodic Acid Schiff’s Combo technique be sued?
Used to separate acid and neutral mucins. Helps in the diagnosis and classification of undifferentiated neoplasms.
What do you expect to stain with an Alcian Blue/PAS combo technique?
- Alcian Blue will stain all acid mucins by ionic bonding –> turquoise blue
- Weak Acid Mucins that are also PAS pos. –> Purple
(i. e. when Harris Hematoxylin is used) - Neutral Mucins –> Magenta
What is Southgate’s Mucicarmine?
Southgate’s Mucicarmine: • Carmine – cochineal beetle (cactus) • Chelated with aluminum salts • Cationic (pos charge) • Binds ionically with epithelial sialomucins (considered specific for epithelial mucins)
What is mucicarmine stain and its clinical purpose?
Stains carboxylated and sulphated mucins, but not neutral mucins.
Specific for epithelial mucins and used to identify adenocarcinomas.
What compounds are used in Mucicarmine stain?
- Mayers (Alum) or Weigert’s (Iron) Hematoxylin –> to stain nuclei
- Mucicarmine –> stains acid mucins
- Metanil Yellow –> counterstain
What bacteria can Mucicarmine be used to stain?
Capsule of Cryptococcus spp. deep rose to red color.
What step in Alcian blue differentiates Hematoxylin? What happens if it is over or under differentiated?
The 0.5% Acid Alcohol performs the differentiation.
Over Diff –> Pale Nuclei
Under Diff –> Bluish colour in background
What is the end result of colours for PAS?
Bright Pink (Magenta) –> Neutral mucins, glycogen, collagen, basement membrane, cartilage, amyloid.
Dark Blue –> Nuclei
Light pink to purple –> all other tissue elements, like cytoplasm, etc.
What do you expect for the end result of a PAS Diastase Slide?
Nuclei - blue
Background - light blue or purple
Carbohydrates that are PAS pos –> Magenta except for glycogen.
What colours do you expect to see with Alcian Blue using Harris Hematoxylin as the counterstain?
Turquoise Blue –> acid mucins, cartilage, mast cells, amyloid
Nuclei –> Blue
Pale –> all other tissue elements
