| 1.
weak signal or no staining |
possible
causes |
suggestions |
-
antibody concentration too low
- inadequate antibody incubation time
-
incomplete deparaffinization
- incompatible primary and secondary antibodies
|
-
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 |
