350 - Special Stains Flashcards
this is used to demonstrate carboxylated and sulfated acid-mucopolysaccharides
alcian blue
alcian blue at pH 2.5
all acid mucosubstances are stained
- both carboxylated and sulfated
alcian blue at pH 1.0
only sulfated mucosubstances are demonstrated
principle of alcian blue
- cationic dye; binds to acids groups of acid mucopolysaccharides
- sialomucins also demonstrated
- at pH 1.0 carboxyl groups are not ionized and therefore cannot react with alcian blue
alcian blue procedure
- NBF or Bouin’s (fixative)
- 3% acetic acid or 0.1 M HCl
> ensures stable pH; acetic acid for 2.5 and HCl for 1.0 - alcian blue dissolved in acid of choice ^
- acid rinse (removes excess alcian blue)
- nuclear fast red or eosin for counterstain
what is demonstrated at pH 2.5 of alcian blue
hyaluronic acid
acid mucosubstances
sialomucins
BLUE
what is demonstrated at pH 1.0 of alcian blue
sulfated acid mucosubstances
LIGHTER blue
control tissue for alcian blue
small intestine
appendix
colon
PAS-Alcian blue
- PAS will demonstrate neutral polysaccharides
- AB before PA oxidation
- tissues with mixture of both = purple
what does congo red demonstrate?
amyloid
- pathological protein; birefringent
- birefringence is considered most specific technique for amyloid
principle of congo red
- amyloid = linear and 2ry protein structures = B pleats
- linear shape of congo red dye fit within the pleats very specifically and at very regular frequencies
- dye molecule held by hydrogen bonding
congo red procedure
- alcohol is preferred fixative
- sections MUST BE cute 7-10 um to demonstrate birefringence under polarized light
- hematoxylin (counterstain)
- wash in running tap water (bluing)
- alkaline salt solution (increases binding sites; reduced background interference)
- congo red (stains amyloid)
colours in congo red
amyloid is deep pink to pale red under brightfield and apple green under polarized light
elastic tissue is pale pink
nuclei is purple blu
T or F. prolonged storage in formalin will decrease staining intensity for congo red
T
it’s also prone to non-specific interations so saturate with salt (ions) to prevent
Crohn’s disease
amyloid
this stains argentaffin substances
and is frequently used to demonstrate melanin
Fontana Masson
other argentaffin substances that may be demonstrated b FM
enterochromaffin cells of small intestine
some tumours
formalin pigment
principle of Fontana Masson
argentaffin reaction
- can bind silver from solution AND reduce that silver to its metallic/visible form
Fontana Masson procedure
- fixative (avoid alcohol if demonstrating argentaffin granules of tumors or enterochromaffin cells)
- warm ammoniacal silver solution (metal impregnation; argentaffin “stained” black)
- gold chloride = toning agent; refines and darkens silver
- sodium thiosulfate (hypo) = fixing agent; removes any unreduced silver
- nuclear fast red or eosin for counterstain
control tissue for Fontana Masson
section of skin as a melanin control (darker skin = more melanin)
section of small intestine or appendix for argentaffin granules
other tisse elements = red to pink
Fontana Masson can be made specific for Melanin
adding a duplicate slide pre=treated with potassium permanganate or H2O2 = will bleach away melanin
- compare; any staining absent on bleached slide = melanin
this demonstrates elastic fibers
Gomori’s aldehyde fuchsin
how is aldehyde fuchsin made?
by adding acidified paraldehyde to an alcoholic basic fuchsin solution
- solution must ripen for 2-4 days prior to staining
T or F. G Aldehyde fuchsin is a progressive elastin stain
T! there is no differentiator used
GAF procedure
- avoid chromate fixatives; formalin preferred
- aldehyde fuchsin to stain elastic fibers
- 70% alcohol rinse to remove excess stain
- light green SF yellowish for counterstain
result of GAF
elastic fibers are purple
other issue elements are green
control tissue for GAF
aorta embedded on edge preferred; skin or muscular artery acceptable
what else will aldehyde fuchsin stain?
beta cell granules of pancreas
sulfated mucins
mast cells
cartilage
all contain abundant negatively-charged substrates, which readily stain using cationic dyes
at what temp must aldehyde fuchsin be stored at?
4C
this stain demonstrates reticular fiers
Gordon and Sweet’s Reticulin
why are silver methods used to demonstrate reticulin
dye methods are not specific or reliable
principle of Gordon and Sweet Reticulin
- induced argyrophilia
- reticulin will not be demonstrated without prior oxidation/sensitization
- oxidation with potassium perm creates aldehyde groups from hexose sugars found in reticulin
- sensitizing metal salt binds to aldehydes => then replaced by silver from diamine silver solution
- silver reduced to its visible metallic form by formaldehyde
Gordon and Sweet Reticulin procedure
- NBF
- potassium perm to oxidize sugars = aldehyde formation
- oxalic acid to remove brown discolouration by potassium perm
- ferric ammonium sulfate = sensitizer; binds to aldehyde; provides sites for silver
- ammoniacal silver solution = impregnation step
- formalin = reduces silver to make it visible
- gold chloride = toner; refines appearance of silver
- sodium thiosulfate = fixer; removes unreduced silver
- eosin or nuclear fast red as counterstain
control tissue for Gordon and Sweet
liver
this demonstrates fungus in tissue sections
Grocott Methenamine Silver
other staining methods for fungus besides GMS
PAS, Gram stain, and H&E
but GMS is most selective for the widest variety of organisms
principle of GMS
- fungal cell walls = lots of polysaccharides
- polysacchss readily oxidized into aldehydes using chromic acid (STRONG oxidizer)
- sections placed in 60C methenamine-silver solution where silver binds aldehydes -> reduce to metallic silver
INDUCED ARGETAFFIN
GMS procedure
- NBF
- chromic acid = oxidizer
- sodium bisulfite - neutralizes residual chromic acid and removes orange
- warm meth silver = impregnation; darkens during this step = remove when tobacco brown; check mic
- gold chloride = toner
- sodium thiosulfate = fixer; removes any unreduced silver
- light green SF (yellowish)
results of GMS
- fungus = black cell walls w grey interior
- mucin is grey
- background = light green
control tissue for GMS
section w fungus
if staining for P. jirovecii, Pneumocystis control needed
What’s so special about P. jirovecii in GMS?
takes longer to stain than other organisms; check microscopically after silver impregnation
also cannot be cultured
this demonstrates connective tissue, esp. collagen
masson trichrome
nuclear stain used in MT
iron hematoxylin b/c alum-mordanted hematoxylin would be differentiated by acidic dyes used in this procedure
principle of MT
porosity is basis of staining
- fixatives react w proteins, changing their shape and formation
- connective tissue more permeable than most other components once they are fixed
- small red anionic dyes will stain components quickly
- PMA or PTA added, which bumps small red dyes from larger pores of collagen; may also act as mordants for larger dyes
- large blue/green anionic dye easily stains porous collagen
procedure for MT
- Bouin’s recommended as picric acid adds additional reactive sites to proteins = intensifies staining
- weigert’s iron hematoxylin = nuclear stain ; wash in running tap water to blue
- biebrich scarlet acid fuchsin = stains everything red; rinse quickly in DH2O
- PMA/PTA = removes red from collagen
- aniline blue or fast green FCF = collagen
- 1% acetic acid = removes excess collagen stain
control tissue for MT
uterus
small intestine
appendix
fallopian tube
modified gram stains demonstrates…
many types of bacteria
modified gram stain procedure
NBF
- crystal violet
- gram’s iodine (mordant)
- acetone, alcohol, or acid-alcohol
- basic fuchsin
- picric acid/acetone = differentiation/counterstain
steps used in the Brown-Brenn modification of the gram stain
red to pink nuclei and yellow tissue elements
this demonstartes neutral lipids or fats
this demonstrates neutral lipids or fats
oil red o
Oil Red O special consideration
must be fresh or frozen; paraffin processing will remove fat from tissues
basis of staining for Oil red O
- selective solubility
- dye is dissolved in propylene glycol or isopropyl alcohol = dye migrates from solvent into any fat present
- as alcohol and xylene will dissolve and fat present = slides must not be dehydrated or cleared
- Aq mounting media used
Oil Red O procedure
- oil red O = stains lipids
- 85% propylene glycol = differentiator; use only when stain is dissolved in PG
- hematoxylin = counterstain; wash in running tap water to blue
PAS is used to demonstrate carbohydrate-containing structures including …
glycogen
neutral mucin
fungus
basement membranes
also reticulin, zymogen granules of pancreas, thyroid colloid, Russell bodies of plasma cells
periodic acid principle
used to oxidize hydroxyl groups within carbohydrates to form aldehydes
Schiff reagent reacts with free aldehydes, binding it to tissue
- subsequent water rinses remove loosely bound sulfurous acid group from the central carbon atom = restores quinoid ring =bright magenta
PAS procedure
NBF or Bouin’s
- periodic acid (weak oxidizer; oxidizes 1,2 glycol groups of several carbs into aldehydes
- schiff reagent = binds to all aldehydes
- rinse with DH2O or sodium metabisulfite = remove excess Schiff
- wash in running tap water = restore quinoid
- Hematoxylin = counterstain
control tissues for PAS
kidney = most sensitive; little carb in basement membrane of glomeruli = easily over-oxidized
glycogen = liver or cervix
why is it important that excess schiff removed before running water step
to prevent non-specific staining
addition of diastase step in PAS
made specific for glycogen
- 2 slides for patient
- prior to oxidation w periodic acid, one slide is treated with diastase ( or salivary amylase) = hydrolyze glycogen into maltose and glucose (washed out w running water)
- if staining present on undigested slide absent on digested slide = must be glycogen
PASD procedure
fixative
- diastase; slides not to be digested remain in DH2O
- periodic acid
- schiff reagent
- rinse w DH2O or sodium metabisulfite
- wash in running tap water = develops colour
- hematoxylin counterstain
control tissue for PASD
cervix more valuable than liver
ectocervix = glycogen which will be digested; endocervix = mucins will not be digested
this is used to demonstrate hemosiderin
Perl’s Prussian Blue
- hemosiderin = yellow-brown, endogenous, within cells made of iron (ferric hydroxide) bound to protein ‘skeleton’
principle of PPB
- true histochemical method
- iron loosely bound will react (hemo- or myoglobin will not)
- acid ferrocyanide used; any ferric iron present reacts to form insoluble blue pigment (ferric ferrocyanide = Prussian blue)
- PPB is not a stain or dye; it’s a chemical rxn
PPB procedure
NBF or alcohol; avoid acid-containing fixatives = may dissolve small iron deposits
- potassium ferrocyanide/hydrochloric acid = reacts with ferric iron forming insoluble blue pigment
- nuclear fast red or eosin for counterstain
results of PPB
blue hemosiderin
pink to red background
why do we rinse slides in distilled water when doing PPB
tap water may contain iron; use distilled water after hydration
asbestos bodies
appear blue in PPB due to ferritin and hemmosiderin content
Verhoeff’s elastic stain is an example of a
regressive stain
Verhoeff proecure
NBF
- elastic stain (hematoxylin, ferric chloride, iodine = stains everything black/dark purple
- ferric chloride = differentiator; elastin will be last to remain stained
- sodium thiosulfate = removes iodine staining
- eosin or van Gieson = counterstain
if van Gieson was used for Verhoeff counterstain = results?
red collagen
yellow muscle/cytoplasm
control tissue for Verhoeff
aorta embedded on edge
skin or muscular artery acceptable
this demonstrates calcium in the tissue
Van Kossa
principle of the VK mehtod
metallic substitution
- silver in stain reacts w carbonate and phosphate groups of calcium carbonate/phosphate
- bright light (sunlight or UV) is used to reduce the silver to visible metallic form
- this method detects anionic groups (not calcium) = indirect method of calcium detection
VK procedure
NBF or alc fixatives
- silver nitrate and sunlight = metal impregnation; silver reacts w carbonate and phosphate salts
- sodium thiosulfate (hypo) = removes nureduced silver
- nuclear-fast red or eosin for counterstain
Alizarin Red S
replaced VK
direct method; dye molecules bind with calcium
principle of ZN
- AFB = mycolic acids = waxy; hydrophobic = prevents staining w most aq stains
- warm carbol fuchsin = alcohol and phenol allow basic fuchsin to penetrate waxy coating of mycobacteria and other bacteria
- decolourize sections with acid alc (1% HCl in 70% ethanol) which acid fast organisms resist ; other tissue elements and bacteria are decolourized
ZN procedure
fixative
- carbol fuchsin
- 1% alc
- methylene blue counterstain
why is millipore-filtered water used on water baths and during reagent prep for ZN?
tap water may contain non-pathological acid-fast organisms
