Shreffler: Hoerscht Counterstaining

Materials

Equipment

• Frosted glass slides, pre-cleaned (Cat. 12-550-343; Fisherbrand)
• Cytospin machine
• Microscope
• Coplin jar

Reagents

• Methanol (Cat. 494437-1L; Sigma-Aldrich)
• Hoescht dye aliquots acquired from Mempel lab (unsure if Hoechst 33258 or Hoechst 33342)

Procedure

1. Cytospin cells onto a frosted slide, pre-cleaned (600 rpm, 5 min, approx. 500 µl cell suspension used).
2. Dry for 15 minutes at RT.
3. Place in 100% methanol for 1 min.
4. Remove and dry for 5 min at RT (keep in dark).
5. Meanwhile, mix Hoescht dye by adding 5 µl of aliquoted stock to 50 ml of diH20 in a Coplin jar.
6. Place slide in the in the Hoescht staining solution in the Coplin jar for 1 min.
7. Wash in diH20 for 5 min without agitation.
8. Dry at RT in the dark for at least 15 min.
9. Analyze by microscopy.