LECTURE - Uncommon Stains: Amyloid & Lipids

Question 1

T or F. processing is detrimental to lipids

Answer 1

T! most routine fixatives will preserve

Question 2

T or F. If lipids are bound, they are more likely to survive

Answer 2

T!

Question 3

this renders lipids black

Answer 3

osmium tetroxide

Question 4

oil red O principle and method

Answer 4

• preferential solubility (soluble in lipids; prefer lipids more than solvents)
• lysochrome: incapable of ionizing = non-polar
• counterstain
• lipids are red

Question 5

cryotomy for staining lipids

Answer 5

• special cutting technique
• freezing medium = external support instead of wax
• decontamination

Question 6

coverslipping for lipids

Answer 6

medium is aqueous becase if using aq. based stain, a non aq coverslip might dissolve away lipids

air bubbles are more prone to occur as well; be careful! might disrupt where lipids are on the slide

Question 7

amyloid

Answer 7

• derived from abnormal protein folding
• insoluble, deposition
• can affect many types of proteins
• dozens of types: AL, AA, ATTR are most common

AL = associated with light chains

• AA = associated with acute phase reactants
• ATTR = associated with transthyretin

Question 8

what is amyloidosis?

Answer 8

• relatively rare but very seirous
• non-functional protein-like entities depositing in organs
• can be primary or secondary
• symptoms related to organs involved
• geographical distribution differences
• histology to diagnose SPE for typing

Question 9

amyloid appearance in stains

Answer 9

• H&E: pink or unstained
• Alcian blue: variably positive
• PAS: variably positive
• MT: can be blue or green
• SPECIFIC STAIN NEEDED

Question 10

Congo red principle for amyloids

Answer 10

• abnormal folding tends to favour B-pleated sheets over alpha helix
• hydrogen bonding is key
• birefringence; use polarized microscope to see as this is a characteristic of amyloidosis
• 2 basic methods to limit non-specific binding = method A and method B

Question 11

method A

Answer 11

• buffered alkaline congo red => change pH, utilize isoelectric pt and manipulate # of charges; limits staining to just amyloid = H bond
• hematoxylin as counterstain

Question 12

method B

Answer 12

• saturated salt solution = BEFORE congo; salt separates + and - and neg matches with their opposites charge in tissues = no space for congo red to join other than amyloid
• congo red
• hematoxylin