This is a Hematoxylin and Eosin (H&E) Staining protocol for mouse colon. For other mouse organs, the immersing time in H&E might be adjusted accordingly.

Procedures

1. Dewaxing

• Incubate the slides under 65 degrees for 30 mins.
• Immerse the sildes in Xylene 1 for 30 mins.
• Immerse the sildes in Xylene 2 for 30mins.
• Immerse the sildes in 100% Ethanol 1 for 10 mins.
• Immerse the sildes in 100% Ethanol 2 for 10 mins.
• Immerse the sildes in 90% Ethanol for 5 mins.
• Immerse the sildes in 70% Ethanol for 1 min.
• Immerse the sildes in 50% Ethanol for 1 min.
• Immerse the sildes in 30% Ethanol for 1 min.
• Immerse the sildes in PBS for 5 mins.

2. Immerse the slides in the distilled water for 5 mins.

3. Stain the nuclei with hematoxylin for 4 mins.

The necessary staining time depends on the tissue type, its fixation method, and the employed hematoxylin.

4. Rinse the slides in running tap water from the reverse side, and rinse the slides in PBS for 5 mins.

5. Stain the nuclei with eosin for 2 mins.

6. Rinse the slides in running tap water from the reverse side, and rinse the slides in PBS for 5 mins.

7. Dehydrate the slides with 100% Ethanol 3 for 2 mins.

8. Dehydrate the slides with 100% Ethanol 4 for 2 mins.

9. Resin the slides in Xylene 1 for 2 mins.

10. Resin the slides in Xylene 2 for 2 mins.

11. Add DPX mountant on the slides, and cover the sample with coverslip.