Staining is too dark — high background obscures cells (Transwell Migration / Invasion Assay)

Question

Accepted Answer

Background is deeply stained; non-specific stain swamps the cells; hard to distinguish migrated cells from background. Common causes: 1) Staining time too long or concentration too high 2) Washing was inadequate 3) Cells on the upper surface were not removed 4) Over-fixation makes everything take up stain Solutions: 1) Reduce staining time (10 – 15 min) 2) Wash thoroughly: 3 – 5 gentle washes in PBS or water 3) Use a cotton swab to gently remove non-migrated cells from the upper surface BEFORE staining 4) Fix for 15 – 20 min only