Primary Antibody: S1antiprotein expensive part

1
General ImmunoHistoChemistry Protocol (with
amplification)
Amplification avidin biotin-enzyme
Reactant
Background Quenching pretreatment with Reactant
Tagging Substrate -gt crystallized product
VISIBLE
Secondary Antibody biotin-S2-anti-S1
Primary Antibody S1-anti-protein (expensive
part)
Background Blocking pretreatment with S2 serum
Target protein in experimental animal (Sx not S1
or S2)
KEY Target Protein protein that is being
identified (e.g. GluR) Sx experimental
species S1 species in which primary antibody
was prepared (e.g. rabbit) S2 species in which
secondary antibody was prepared (e.g.
goat) Enzyme amplification enzyme (e.g.
HRP) Substrate amplification product (e.g.
DAB) - binding reactions
2
Specific ImmunoHistoChemistry Protocol (with
amplification)
Amplification avidin biotin-HRP
(HorseRadish Peroxidase)
H2O O?
VectorLabs ABC Kit ( 250 / 1000 sections)
H2O2
Ni
DAB DiAmidinoBenzidine (Carcinogenic part)
VISIBLE Crystal
Quenching H2O2
Secondary Antibody biotin-Goat-anti-Rabbit
Primary Antibody Rabbit-anti-GluR (expensive
part)
Blocking Goat serum (in kit)
Rat - GluR
Goat Serum blocks non-specific binding of the
secondary antibody to rat proteins Peroxide
quenching deletes all endogenous peroxide
activity (presoak anytime before amp)
Diamidinobenzidine crystallizes by oxidation from
free radicals generated by peroxidase
3
General Protocol for VectorLabs ABC Kits
http//www.vectorlabs.com/infopage.asp?dpID4locI
D609308
4
Multiple Labeling (i.e. one protein brown,
another red) is accomplished by repeating
protocol using different enzyme substrate
http//www.vectorlabs.com/infopage.asp?dpID27loc
ID609308
5
VectorLabs supplies enzyme substrates for several
multi-label contrast options
Estrogen receptor (brown, DAB), endothelium
(blue, Vector Blue), epithelium (red, Vector Red)
http//www.vectorlabs.com/infopage.asp?dpID30loc
ID609308