| 1. weak signal or no staining | |
| possible causes | suggestions |
- antibody concentration too low | - increase antibody concentration - increase antibody incubation duration - increase toluene (xylene) immersion time; if toluene has been used many times, replace with fresh solvent - use secondary antibodies that target the species that the primary antibodies were raised in |
| 2. high background signal | |
| - secondary antibody too concentrated - inadequate washing stringency - sections dried during procedure - endogenous peroxidase | - decrease secondary antibody concentration - increase PBS washing steps or washing duration - always work quickly to prevent tissue sections from drying - use endogenous peroxidase quenching buffer |
| 3. tissue sections fall off microscope slides | |
| - excessive ajitation during PBS washing steps - microscope slide surface not binding effectively to tissue sections | - decrease PBS washing duration - use charged microscope slides or treat slides with poly-L- lysine |
| 4. overstaining of tissue sections | |
| - primary antibody concentration too high - primary antibody incubation too long - non specific labeling | - decrease primary antibody concentration - decrease primary antibody incubation duration - use a blocking agent such as normal serum or bovine serum albumin |
| 5. tissue section morphology has deteriorated appearance | |
| - incomplete or lacking tissue fixation - overdigestion with permeabilization solution | - perform tissue fixation protocol - decrease Triton X-100 concentration or duration of incubation in permeabilization solution |