Aclian Blue Stain Flashcards
What is the target of alcian blue
Mucin
carbohydrate group
mono - simple sugar
poly-
muco polysacc - long chain of sugars found in the body , needed for cell growth and structure
What is part of group 1 poly sacc
-carry no charge
Neutral poly (non ionic homoglycans)
-found in stomach and bruners glands
-Glucose containing glycogen, starch and cellulose
N-acetyl -glucoasmine containing CHITIN
PAS positive
AB negative , Collodial iron negative, Mayers mucicarmine method MMM positive
What is part of carbohydrate group 2
Acid mucopolysaccharides (anionic heteroglycans) CARRY A NEGATIVE CHARGE ON MUCIN MOLECULE
Classifed as simple with
Carboxyl group- hyaluronic acid (connect tissue and umbilical cord)
Complex
Sulfuric group and Carboxy : chondroitin A, B, C (cartilage, cornea, blood vessels, aorta) and Heparin
Sulfated : Aorta
All acidic and attached to proteins
Pas NEGATIVE
AB positive
Colloidal iron POSITIVE
MMM Positive
Group 3 carbs consist of
Glycoproteins (mucins, mucoid, mucoprotein, mucocutaneus )
mainly epithelial mucins
neutral - stomach mucin , paneth cells
Carboxylated - sialoglycoprotein
Sialomucins - fetal mucin , upper part of crypts
Sulfated and Caboxylated - colonic mucins in goblet cells
PAS - POS
AB - POS
Group 4 carbs are
Glycolipids
Cerebrosides - lipid attached to carbohydrates
phosphatides - non carb containing lipids
PAS - POS
What are the stains that demostrate mucins
MMM
AB
PAS
Colloidal Iron
What is the purpose of alcian blue stainings
-demonstation of acid mucins (mucopoly) and sialomucins (glycoprotein)
-group 2 and 3 carbohydrates
-bind with ionic bonding or salt linkage
What are the control tissues for AB
Positive control - small intestine and appendix ANY GI
if ABPAS is used - duodenum is the best tissue because the brunners gland with neutral poly that pick up the PAS staining
always in include a small section of the kidney - most sensitive control for PAS
What are the characteristics of AB
-powder
-has copper phthalocyanin - basic dye with positive charge
-water soluble
-blue color because of the copper
how is the AB solution made
-powder dissolved in 3% acetic acid pH 2.5
-demonstrates sulfated and caboxylated acid mucopolys and sulfated/carboxy sialomucins
-if its made to pH 1.0 itll be ONLY sulfated acid muco and sulfated sialomucin
What are the advantages of Alcian blue over other cationic dyes
water, alcohol, weak acids and other counter stains will not extract the dye from stained sections
-the dye is strong
-incubaction time is adjustable
-dye can be combined with PAS/Hx to demonstrate neutral mucins AND acid mucins
What is ionic bonding
bond between cation and anion
staining depends on the charge of the dye and the charge of the tissue
-opposites attract
Acid dyes ANIONIC are _____ they bind to ___charged tissues and the tissue is described as
Acid dyes are NEGATIVE they bind to POSITIVE charged tissues and the tissue is described as ACIDOPHILIC
BASIC - CATIONIC dyes are _____ they bind to ___charged tissues and the tissue is described as
BASIC - CATIONIC dyes are POSITIVE they bind to NEGATIVELY charged tissues and the tissue is described as BASOPHILIC
What are amphoteric dyes
both basic and acidic groups behaving as both above or below a certain pH known as a isoelectric point
at pH 4 stains cytoplasm (positive charged) so it is an acid dye at pH 4 or below
protein tissue can also be amphoteric - 6-6.5pH
below the isoelectric point it is acidophilic or positively charged and above the pH its basophilic
How would protein staining on an acid solution work
if acid dye is negatively and is put on a protein in an acidic enviroment in low pH = BRIGHT Color because there are more reactions between the dye and tissue
as pH increases the intensity is weaker
If Basic dye is applied to protein in acid environment = weaker color and as pH goes up the intensity of the color goes up
How would protein staining work in a basic solution
Basic dye is more intense in basic solution
Acid loses intensity as the solution gets more basic
What happens if Alcian blue is added to tissues (cryts of lieberkuhn CL ) at different pH
1- little blue reaction
2-stronger
3-stronger
4-6 no staining in CL but other cells were stained
7 and up - no staining anywhere
AB does not stain mucipoly at pH of 4 or up
Acts as a basic dye at pH 1-4 , acid dye at pH 4-7 = amphoteric dye
Why is there a 2nd 3% acetic acid rinse in AB staining
prevent non specific staining
dont want to change pH while stain is on the slide so under acetic acid before rinsing in tap water
What is the step 1 of AB staining
acetic acid rinse
-same solvent as alcian blue solution
-protects AB staining solution from pH changes after being exposed to water
Step 2 of AB staining
Staining
-done at pH 2.5 or 1.0
-amphoteric dye
-progressive stain and direct
-binds to negatively charged mucopoly and sialomucins which are caroxy/sulfated at pH 2.5 and sulphated at pH 1.0 (if pH 1.0 ordered then instead of acetic acid use HCL acid)
-if ABPAS because the neutral carbs have no charge they wont stained
-creates insoluble end product
step 3 of AB staining
Counter staining
-ionic bonding
-demonstrates other tissue components
-has to be a contrasting color
-diff counter stains can be used
Hx, PAS, NFR, VG
-Most common is Nuclear Fast Red
What would be a result of the staining
Expect: Mucin stained and counterstain should enhance the mucin
@ph 1.0
sulphated acid mucopoly and glycoproteins - pale blue
Background - pink to red
@pH 2.5
Weakly acidic mucopoly, hyaluronic acid and sialo - dark blue
background - pink to red
pg 33 what ph
2.5
Nuceli are red
34
ph 1
35
ph 2.5
37
neutral mucins vs acid mucin
2.5 (acid) first then PAS stain (neutral)
duodenum
brunners gland top left
