1. Prolonged incubation or high antibody concentration may increase background staining; shorten primary/secondary antibody incubation and dilute the antibody. 2. Polyclonal primary antibodies tend to cause non-specific staining; monoclonal antibodies are recommended. 3. Endogenous peroxidase and biotin are abundant in liver, kidney and other blood-rich tissues; extend inactivation time and increase inactivating-agent concentration. 4. Non-specific components bind to antibodies; extend blocking with serum from the secondary-antibody host species and increase its concentration. 5. Excessively long DAB incubation or high DAB concentration increases background; reduce DAB concentration or shorten incubation. 6. Insufficient PBS washing leaves residual antibody and strengthens staining; wash thoroughly after primary antibody, secondary antibody and SP incubation. 7. Dried sections during staining often enhance non-specific staining; keep sections moist throughout. 8. Low pH of the antibody diluent or expired blocking serum.
Technical Support
Technical notes on IHC staining issues and troubleshooting.
1. Specimen fixation: improper fixation or excessively high temperature reduces antigen quantity and quality; follow the primary-antibody datasheet and use the recommended fixative (e.g. 10% neutral buffered formalin). 2. Antigen retrieval: heat retrieval and pressure-cooker retrieval are widely accepted; choose the method based on preliminary tests or the manufacturer’s instructions. 3. Antibody dilution may be too high, or incubation temperature/time incorrect; titrate the primary antibody to find the optimal working concentration, and extend incubation if background is low. 4. Excess wash buffer left on the slide further dilutes the antibody when it is applied. 5. Place slides horizontally during incubation, otherwise antibody may run off.