An
Autostainer XL (ST5010, Leica Biosystems, Nussloch, Germany) was used to
perform standard H&E staining. The automated staining protocol consisted of
the following steps: three changes of xylenes (2, 2 and 1 minutes), rehydration
in an ethanol/water gradient (100%, 100%, 95% ethanol for 2, 1 minutes and 20
seconds, respectively), followed by washing with deionized (DI) water (2 minutes).
The rehydrated slides were stained with Gill 3 Hematoxylin (12 minutes;
Sigma-Aldrich), washed with DI water (2 minutes), followed by 0.17% Acid
Alcohol (1 minutes), washed with DI water (1 minutes), Ammonia water (20
seconds), washed with DI water (1 min), and 95% ethanol (10 seconds). Staining
with Eosin (45 seconds; Sigma-Aldrich) was completed, followed by four washes
with 100% ethanol (1 minute each), and four washes with xylene (1 minute each).
Lastly, H&E stained slides were cover slipped with Cytoseal XYL (Thermo
Fisher Scientific) and allowed to dry flat prior to imaging.