Key points of quality control and common problems in the whole process of immunohistochemical experiments
Sample Fixation and Handling: The Starting Point for Stable IHC Results
The reliability of immunohistochemical (IHC) results depends first and foremost on the pretreatment of tissue samples.Common specimens include paraffin-embedded tissue and frozen sections.The paraffin tissue should be fixed as soon as possible after ex vivo, and the type of fixed solution, fixed time and temperature will affect the antigenic activity.Inadequate immobilization may cause antigen dispersion, and excessive immobilization may cause antigen epitopes to be obscured.It is generally recommended to use a neutral buffer formalin and control for a fixed period of time according to a standardized laboratory process.Dense specimens such as bone tissue need to be decalcified, and the decalcification time also needs to be balanced between antigen preservation and morphological integrity.
Slice thickness, slice temperature and storage conditions are also noteworthy.Too thick slices can easily lead to enhanced background staining, and too thin slices can lead to weak signals.Long-term storage of slices should avoid high temperature and humidity to avoid further degradation of antigens.For special indicators, it is recommended to determine the optimal slicing conditions according to the antibody instructions and internal verification results.
Antigen Repair and Antibody Selection: Core Links Affecting Specificity
Antigen repair is a key step in restoring antigenic epitopes in paraffin sections. Common repair methods include heat-induced repair and enzymatic digestive repair.Thermal repair usually uses citrate buffer or EDTA buffer. Different antibodies are sensitive to the pH value of the repair solution, which needs to be determined by pre-test.False negatives can occur if repairs are inadequate, and excessive repairs can disrupt tissue morphology and increase nonspecific shading.
Antibody selection and dilution directly determine the quality of staining.