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G-Banding - Its Applications in Cytogenetics

BY: SUNIL KUMAR, S.V. | Category: Agriculture | Submitted: 2012-03-29 22:26:38
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Article Summary: "Chromosomes are G-banded to facilitate the identification of structural abnormalities. Slides are dehydrated, treated with the enzyme trypsin, and then stained..."


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Principle
Chromosomes are G-banded to facilitate the identification of structural abnormalities. Slides are dehydrated, treated with the enzyme trypsin, and then stained.

Equipment
Glass coplin jars with lids; 5ml and 10ml serological pipettes and bulbs; Staining rack; Timer; Plastic squirt bottle; Dehydrating oven set at 95 degrees C; Slide racks; Slide warming tray set at 60 degrees C;

Reagents
0.25% trypsin, in HBSS without calcium and magnesium. (Life Technologies Inc., Gibco #610-5050) Store at -5 to -20 degrees C. Thaw at room temperature -- do not heat above room temperature to avoid denaturing the enzyme.

Dry salt phosphate buffer, pH 6.8 (Fisher Scientific #B-78)
Leishman's stain powder. (Sigma #L-6254)
Absolute methanol. (J.T. Baker, VWR #JT9070-3) Toxic -- avoid contact with skin and mucous membranes. Flamable -- store in approved fire-proof cabinet. Dispose down fume hood sink with copius amounts of water.

Normal saline, 0.9% sodium chloride. (Abbott Laboratories, SHMC #3619)
pHydrion buffer capsules, pH 7.0 (VWR #34175-242)

0.025% trypsin: mix 5ml 0.25% trypsin with 45ml normal saline. Prepare fresh the day of staining. Keep at room temperature. Do not use if color changes.

Fisher phosphate buffer: dissolve 1 buffer capsule (pH 6.8) in 1 liter of distilled water. Check pH with pH meter and record on the label.

Stain: swirl 500ml absolute methanol in flask. Add 1.0g powdered stain to swirling methanol. Continue to swirl 2-3 minutes at moderate rate. Let sit for 15 minutes. Filter through Whatman #1 filter paper into brown bottle that contains no water (swirl bottle with methanol before use). Store away from heat and light. Shake well and filter before use.
pHydrion stock buffer: dissolve 1 pHydrion buffer capsule (pH 7.0) in 100 ml distilled water. Check pH with pH meter and record on label. Store at 2 to 8 degrees C.
pHydrion working solution: mix 5ml pHydrion stock buffer with 95ml of distilled water. Store at room temperature.

Procedure
1. Place fixed, dry slides on slide rack in 95 degrees C oven and bake for 20 minutes. Cool.
2. Immerse slide in 0.025% trypsin for 10 to 120 seconds.
3. Remove slide from trypsin and immediately immerse in Fisher phospate buffer to stop trypsin action.
4. Place slide cell side up on staining rack and flood with solution of 1 part Leishman's stain and 3 parts pHydrion working solution. Stain for 2 minutes.
5. Rinse slides thoroughly with distilled water.
6. Allow slides to drain, then place on 60† C slide warming tray until completely dry.

Notes and Precautions
1. Trypsinization time may need to be varied depending on environmental conditions, material being banded, or typsin stock. Always stain one slide first and check banding quality before staining additional slides. Adjust trypsinization time if necessary.
2. Be careful not to over rinse slides since over rinsing will fade stain.
3. Leishman's stain will form a precipitate when added to pHydrion buffer. Therefore, the two should not be mixed until just prior to flooding the slide.
4. Step 3 (Fisher phospate buffer) may be eliminated if desired.
5. Slides can be destained by placing them in a coplin jar containing 3:1 fixative (3 parts absolute methanol mixed with one part glacial acetic acid) for 1-2 minutes. Rinse slides with absolute methanol and allow to dry completely. Slides may then be re-trypsinized and/or restained.
6. Wright's stain may be used in place of Leishman's stain.

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