H&E Staining of Paraffin-Embedded Tissue Sections: A Differential Staining Technique to Visualize Liver Tissue Sections Using Combination of Hematoxylin and Eosin Dyes

Deparaffinize the slides by immersing them in 100% xylene, twice, for 15 minutes each time. Then, rehydrate the slides through an ethanol gradient. For each solution, dip 8 to 10 times, for two seconds per dip, until the liquid runs cleanly off the slides. Place the slides in 100% filtered Harris hematoxylin for four minutes.

After four minutes, quickly transfer them back to the container of deionized water. Run deionized water into the back corner of the container farthest away from the sections. Empty the container periodically until the water is no longer purple.

Quickly check the hematoxylin intensity on a dissecting microscope using gooseneck lights. Return the slides to 100% hematoxylin for one minute, if further staining is required.

Once the desired hematoxylin staining has been obtained, dip the slides twice in 0.05% hydrochloric acid, then, immediately transfer them back to the container with clean deionized water. Empty and refill the container with water twice.

Transfer the slides to two containers of 95% ethanol for 30 seconds each. Place the slides into the eosin Y phloxine-B solution for two minutes.

After the two minutes, transfer the slides back to the previous 95% ethanol container, then check the intensity of the color under the dissecting microscope. If the staining is not sufficient, return to the eosin solution for 30 seconds. Repeat as necessary.

When staining of the desired intensity has been obtained, transfer the slides to 100% isopropanol for 15 seconds. Replace with fresh 100% isopropanol, and place the slides back in the isopropanol for another 15 seconds. Repeat this process for a total of six isopropanol washes.

After immersing in 100% xylene for three minutes, remove one slide, add sufficient mounting medium to cover the sections, and then, dip the slide in 100% xylene.

Apply a coverslip to the slide and blot any excess mounting medium on a paper towel until only a thin line is seen. Then, dip a tissue into the xylene and wipe the back of the slide to remove any medium that has dripped.

Place the slide flat on a sturdy but mobile surface, like a piece of cardboard, and allow the xylene to evaporate in the hood for 10 minutes. Leave the slides at room temperature overnight to allow the mounting medium to harden before imaging.