Unexpected staining patterns
Unexpected staining patterns are commonly due to suboptimal digestion conditions. Generally speaking, over-fixed or under-digested tissue will have excellent tissue morphology with weak/no signal and low signal/background ratio due to poor probe accessibility to RNA.
Under-fixed or over-digested tissue will have poor tissue morphology (tissue appears faded with loss of cell borders) and loss of RNA due to protease over-digestion. Use below examples of unexpected staining patterns to determine if your sample is under- or over-digested.
