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Perform this procedure under a chemical fume hood. Wear two layers of nitrile gloves. Lab coat and eye protection required.
Hazardous chemicals used in this procedure: acetic acid, xylene, DPX
- In 0.9% NaCl (aq), spread out vibratome sections of the perfusion-fixed brain in sequence from rostoral to caudal (refer to the rat brain atlas).
- Mount the sections in sequence onto glass microscope slides.
- Air dry the mounted sections 2-3 days.
- Load staining rack with slides with the sections.
- Prepare staining dishes: purified water, Cresyl Violet, 100% ethanol, 100% ethanol with 0.1% acetic acid (glacial), 100% ethanol × 4, xylene × 3
- Place the rack containing slides in purified water, 2 min
- 100% ethanol × 2, 3 min each
- Xylene, 4 min
- 100% ethanol × 2, 4 min each
- 95% ethanol, 4 min
- 75% ethanol, 4 min
- Purified water × 2, 1 min each
- Drain and briefly dry the sections
- cresyl violet solution, 2 min
- Rise briefly in 100% ethanol
- 100% ethanol with 0.1% acetic acid (glacial), ~1-2 min
- 200 ml working vol EtOH, 200 µl Acetic Acid
- 100% ethanol × 4, 2 min each (check staining with a microscope after 2 changes)
- Xylene × 3, 3 min each
- Coverslip with DPX mountant - keep slides in xylene until coverslip is applied
- List of slides:
- Let dry in hood ~48 hrs