All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Staining
- Before proceeding with staining, wipe off any excess solutions around the tissue on the slide with tissue paper.
- Place the slides in room temperature and room humidity conditions to avoid drying of the tissue on the slide throughout the entire staining process.
- Oxidize the slides with a few drops of potassium permanganate for 5 min. Ensure that the volume of potassium permanganate is enough to cover the tissue section on each slide.
- Rinse the oxidized slides with distilled water in a Coplin jar with 2-3 changes. Observe the blue color of the tissue.
- Now, bleach these slides by adding a few drops of oxalic acid for 5 min. Ensure that the volume of oxalic acid is enough to completely cover the tissue section on each slide.
- Rinse the slides by immersing them in the Coplin jar filled with distilled water. Perform the rinse 2x-3x.
- Now, wash these slides briefly in 95% alcohol, and then immerse the slides into an elastin solution (5 g/L) for 8-24 h.
NOTE: Elastin solution is volatile, and its staining time is relatively long, so it is best to immerse the slides with elastic staining in a sealed container, such as a transparent glass bottle with a sealing cap. The capacity of the container and the volume of the elastin solution depends on the number of immersed slides, as long as it enables the complete immersion of all the slides.
- Immerse these slides directly in 95% ethanol for 1-2 min to differentiate each tissue section well.
NOTE: Differentiation refers to the change in the charge of the tissue section, which removes the excess staining absorbed by the tissue and makes the colors clearer. After elastin solution staining, samples should not be washed with distilled water to avoid any difficulty in differentiation. Check microscopically, as necessary, for blue-black elastic fibers staining and gray background. It is better to slightly under differentiate the tissue since the subsequent Van Gieson's counterstain can also somewhat extract the elastic stain.
- Fully rinse these slides with distilled water after differentiation.
- Counterstain the slides in Van Gieson's solution for 1 min.
NOTE: The volume of Van Gieson's solution needs to be enough to be able to immerse the tissue section on each slide completely.
