Giemsa-trypsin Banding

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Giemsa-trypsin Banding

Giemsa-trypsin Banding
Section of Cancer Genomics, Genetics Branch, NCI
National Institutes of Health

Reagents
Earles Balanced Salt Solution (BSS)
Fetal Bovine Serum
Giemsa stain, original azure blend
Harleco 620G/75
Gurrs buffer tablets, pH 6.8
Biomedical Specialties, Santa Monica, CA
Trypsin solution 2.5%
GIBCO, Cat. 15240-062
Water, sterile

Preparation
Trypsin working solution
Trypsin solution, 2.5% 1.5 ml
Earles BSS 50 ml

Fetal bovine serum solution
Fetal bovine serum 1 ml
Earles BSS 50ml

Gurrs buffer solution, pH 6.8
Dissolve 1 tablet in 1 L sterile water

Giema Stain working solution
Giemsa stain 1 ml
Gurrs buffer solution, pH 6.8 50 ml

Procedure
1. Age the air-dried slide overnight in a dry oven at 55-60°C. Remove and bring to room temperature just prior to banding.

2. Grasp slide with forceps and dip in trypsin solution. Use a back-andforth motion and expose to solution for 8-10 seconds. Time may have to be adjusted, depending on the degree of banding.

3. Quickly remove slide from trypsin and dip in fetal bovine serum solution. Move slide back-and-forth in the solution, or dip 5 times.

4. Pre-rinse the slide by dipping in a third jar containing Gurrs buffer, using the same technique as in step 3.

5. Place slide in Giemsa staining solution for 8-10 minutes.

6. Rinse slide in distilled water, using the same technique as in step 3. (If slide is left in water too long, the stain will fade).

7. Allow slide to air dry. Examine without mounting, using dry objectives; coverslip with Permount for examination with oil objective.

Link: http://www.riedlab.nci.nih.gov/publications/2455.2133_Giemsa_Trypsin_Banding.pdf