underpermeabilization
Underpermeabilization is a term used in immunostaining to describe insufficient permeabilization of cells or tissue, which prevents antibodies from accessing intracellular epitopes. It typically manifests as weak or absent staining for intracellular targets while surface antigens may still be detectable. Permeabilization is usually achieved with detergents such as Triton X-100 or NP-40, or with alcohol-based methods like methanol; the choice depends on sample type and the epitopes of interest. Inadequate permeabilization can result from too low detergent concentration, too short incubation, low temperature, or the use of fixatives that overly crosslink membranes (for example, high concentrations of glutaraldehyde) which reduce membrane permeability. Cell type and tissue architecture also influence permeabilization efficiency; dense cytoskeletal networks, thick tissue sections, or heavily crosslinked samples are more prone to underpermeabilization.
To address underpermeabilization, protocols may be adjusted by increasing detergent concentration or incubation time, trying alternative