Congo Red Flashcards
What is congo red classified as
target
staining principle
mechanism of staining
classified as : Carb stain
target: amyloid
staining principle: hydrogen staining
mechanism of staining: non ionic bonding
Fixatives for CR
Alcohol
Carnoy solution
the preferred because prolonged storage in 10% NBF will decrease the intensity of staining. Most use 10% NBF
10% NBF
Bouins solution
Zenkers Solution
Technique for CR
Sections have be cut at 8-10 um
-thin sections may not show the apple green birefringence
-use polarizing microscope to see
What is the QC used for CR
-any tissue with amyloid (kidney or prostate)
-prolonged staining can diminish staining
-dont cut too many controls as the sections get older the staining intensity decreases
-long standing amyloid deposits have a lesser reaction than small newly formed ones
-large deposits show less intense staining
staining procedure for CR
TWO working solutions A (alkaline treatment) leave in for 20 mins and use this time to prep B
DONT RINSE WITH WATER JUST DRAIN
Solution B is made and used within 20 mins
DONT rinse with water
flood slides
drain
DONT rinse
DCM
What stock and working solutions
Stock 80% alco with NaCl
working Solution A = 50 mL stock and 0.5ml 1% NaOh
Stock CR
Solution B- stock CR 50 mL and 0.5 mL 1% NaOH
What is step 1
Nuclear staining
-counterstaining
Harris HX
-progressive
-shouldnt compete with 1ry stain
-stains the nuclear portions but is acidified so any non specific background staining is reduced
-does not need differentiation
Glacial acetic acid
Step 2
Alkaline pretreatment in solution A
-80% alcohol, NaCl, NaOH
-high pH of solution increases staining specificity
-salt solution
-reduces background staining because most proteins will not stain with an anionic dye at this pH
-solutions with electrolyte and organic solvent will inhibit ionic bonding
Step 3
Amyloid solution B
-progressive staining
-permanent
-collagen and elastic can also be stained with CR thats why you pretreat is done because you only want the amyloid to be stained
-selectivity of this stain is due to amyloid composition and contents of the staining solution
What does the amyloid molecule look like
Linear
-antiparallel configuration that makes it easier for hydrogen bonding between tissue and dye
What is hydrogen bonding
-when hydrogen is weakly attracted to O, N or F atom
-CR is made up of alcohol because water form H bonds and it would get in the way of H bonding between tissue and dye
What should be seen in a good CR stain
- Amyloid - salmon pink
-stain intensity depends on size, density, age of amyloid deposit
-Yellow orange fluorescence but this has low specificity so it needs to be confirmed with a polarizing microscopy
-seen as birefringence
elastic fibers - pale pink
nuclei - blue
physical properties of amyloid
Light micro - H/E staining - amorphous eosinophilic deposits
Polarizing light - CR staining - apple green birefringence
EM - Regular fibrillary structure
Xray diffraction - B pleated sheets
What does Amyloid look like under a microscope
Fibrils -10 um
H/E amorphous eosniophilic extracellular fiber
PAS = weak + due to carbohydrates
AB= pos due to sialic acid in glycoprotein
MTC- same color as collagen
Autofluorescent - Weak yellow orange under fluorescent mic
Birefringence - YEs under polarizing light
Other Amyloid diagnositic technique
PAS and AB
Metachromatic stain
Fluorochrome thioflavin T
IHC
PAS and AB- poor dye uptake
Metachromatic stain- crystal violet and methyl violet - lpw specificity and low sensitivity
Fluorochrome thioflavin T- stained sections are not permanent - low specificity
Excitation and barrier filters might not be in most labs
Yellow orange fluorescence
IHC- not all AB are available
CR for diagnostic and confirmatory stain for amyloid
