Cresyl Violet Staining
JLJ July 2016
Solutions Needed
Cresyl Violet Stain (0.1% Cresyl Violet Acetate)
70% Ethanol
95% Ethanol
100% Ethanol
Xylene
Acid Alcohol: 95% Ethanol + 4-5 drops of 10% Glacial Acetic Acid
Cresyl Violet Stain (500 ml)
0.5 g Cresyl Violet Acetate (Sigma C5042)
1.25 ml Glacial Acetic Acid
to 500 ml with distilled H2O
Heat solution to 60 C with constant stirring until majority of crystals are dissolved. If possible, stir solution for
several days with heat. Let the solution cool and store in dark bottle. Reheat to 60 C and filter before every
use. If you see specks of dye on stained sections, use a 0.45 m bottle top filter rather than a fluted paper for
better filtration.
Procedure
Mount sections on subbed or Superfrost Plus slides. Air-dry at room temperature overnight or up to several
days.
Set up the alcohol series: two 70% ethanol, four 95% ethanol, four 100% ethanol and four xylene dishes.
Rehydrate sections under lukewarm tap water for 2-3 min.
Delipidate sections through alcohol series into xylene with 4-5 dips in each jar:
H2O, 2x 70%, 3x 95%, 4x 100% ethanol, 4x xylene.
Rehydrate sections from xylene back to water, going backwards through the alcohol series.
Place slides into heated (60º C), filtered Cresyl Violet Stain for ~ 3-4 minutes or until the stain is slightly too
dark.
Rinse off excess stain in running tap water.
Begin dehydration through 2x 70% and 3x 95% ethanol.
Differentiate the stain by alternating between 95% ethanol and acid alcohol 95% until the background clears
but the cells remain dark purple.
Finish dehydration through 100% ethanol to xylene.
Coverslip with Permount and let dry overnight.