What are the causes of weak staining in tissue sections and how to avoid it?

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.