An immunohistochemical stain may show excessive background staining if certain conditions are present. Identifying the cause can suggest the appropriate solution.
Potential causes for high background staining:
(1) precipitate formed by antibody aggregates
(2) excessive antibody concentration
(3) binding of antibody to Fc receptors in tissue
(4) nonspecific binding of reagent antibody
(5) inadequate washing
(6) inadequate blocking of antibodies in tissue to species used to develop reagent antibody (antimouse antibodies to antibody produced in mouse myeloma cells. etc.)
(7) nonspecific antibodies in reagent that react with tissue antigens
(8) slide dried out during immunostaining
(9) incubation conditions (incubation period to long or incubation temperature too high)
(10) inadequate inhibition of endogenous enzymes (peroxidase, alkaline phosphatase)
(11) edge effect (gap between outer edge of tissue section and slide)
|
Potential Cause |
Solution |
|
precipitate with antibody aggregate |
centrifuge or filter reagent to remove precipitates |
|
excessive antibody concentration |
determine optimum working dilution |
|
binding to antibody Fc receptor |
use F(ab)2 with no Fc component |
|
nonspecific antibody binding |
increase washing steps and increase length of incubation period with normal serum from same species as that used to produce the reagent antibody |
|
inadequate washing |
improve washing steps |
|
inadequate blocking of host antibodies to species used to produce the reagent antibody |
increase length of incubation period with normal serum from same species as that used to produce the reagent antibody |
|
nonspecific antibodies in reagent antibody mixture against tissue species |
absorb reagent antibody mixture with serum from same species as tissue source |
|
slide dried out |
keep the section from drying out |
|
incubation too long or too warm |
optimize incubation conditions |
|
inadequate inhibition of endogenous tissue enzymes |
in a peroxidase system use new hydrogen peroxide; in a alkaline phosphatase systems use levamisole |
|
edge effect |
use slide adhesive to firmly attach sections |
Specialty: Clinical Laboratory
