An immunohistochemical stain may show negative or weak tissue staining if certain conditions are present. Identifying the cause can suggest the appropriate solution.
Potential causes for negative or weak tissue staining:
(1) epitope targeted by the antibody lost during tissue collection, storage or processing
(2) epitope present but destroyed during antigen retrieval process
(3) epitope present but not accessible to antibody (masked)
(4) antibody inactivated or contaminated
(5) antibody concentration too low or very high (prozone)
(6) incubation period with antibody too short
(7) incubation conditions suboptimum (interfere with antigen antibody reaction)
(8) enzyme activity for test signal negative or weak
(9) correct substrate or conjugate for enzyme system weak or inactive
(10) incorrect substrate or conjugate for enzyme system
|
Potential Cause |
Solution |
|
epitope lost during tissue handling |
change tissue handling |
|
epitiope destroyed during antigen retrieval process |
select optimum antigen retrieval procedure |
|
epitope masked |
select optimum antigen retrieval procedure |
|
antibody inactivated |
use positive control |
|
antibody concentration not optimum |
determine proper antibody concentration |
|
incubation period too short |
determine the optimum incubation time |
|
incubation conditions suboptimum |
determine the optimum incubation conditions |
|
enzyme activity negative or weak |
test enzyme activity; make sure there are no enzyme inhibitors present |
|
substrate or conjugate weak or inactive |
test substrate or conjugate for activity before use; use only active material |
|
incorrect substrate or conjugate |
select correct substrate or conjugate |
If potential causes of a false negative reaction have been excluded or corrected, then a weak or negative reaction indicates absent or weak concentration of the target epitope (true negative reaction).
Specialty: Clinical Laboratory
