Micromount

Frozen 7 μm cryosections from healthy and psoriatic lesional skin tissue, stored at −80°C, were thawed at room temperature for 10–20 min and hydrated in PBS for 5 min. They were stained with H&E(Vector Laboratories, H‐3502) according to the manufacturer's instruction. Briefly, thawed and hydrated sections were completed immersed in Hematoxylin for 5 min followed by two changes rinse in distilled water. Bluing reagent was applied for 10–15 seconds, again followed by two changes rinse in distilled water. The slides were dipped in 100% ethanol for 10 s and incubated for 3 min in Eosin Y solution, followed by a rinse and dehydration in 100% ethanol. The slides were mounted with a xylene‐based mounting medium (Micromount, Leica Biosystems, 3801731).

For IHC, four-micron sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost Plus slides and subjected to IHC using anti-nucleocapsid rabbit polyclonal antibody (3A, developed by our laboratory) with the method previously described (80 (link)). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems). Following automated deparaffinization, heat-induced epitope retrieval (HIER) was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody [anti-SARS-CoV-2 nucleocapsid rabbit polyclonal antibody diluted at 1:5,000 in primary antibody diluent (Leica Biosystems)] for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 min), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60°C oven for 30 min and mounted with a permanent mounting medium (Micromount, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

For IHC, 4 μm sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost® Plus slides and subjected to IHC using an anti-nucleocapsid rabbit monoclonal antibody (HL344, Cell Signaling Technology, Danvers, Massachusetts). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems, Wetzlar, Germany). Following automated deparaffinization, heat-induced epitope retrieval (HIER) was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody (diluted at 1:1,600 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 minutes), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60 °C oven for 30 min and mounted with a permanent mounting medium (Micromount®, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

Fish abdominal regions were cut and fixed in formalin solution (Sigma-Aldrich), dehydrated and embedded into paraffin. Sections at 5 μm thickness were processed using a microtome. These sections were deparaffinized, rehydrated and stained with Mayer’s hematoxylin (Vector Laboratories) and eosin (Sigma-Aldrich). The stained slides were mounted in Micromount (Leica) and imaged using an inverted light microscope (Zeiss, Axiovert 200 M). Classification of tumor types were based on established criteria as previously reported34 (link). For hyperplasia, the major criteria are maintenance of hepatic plates, large nuclei with occasionally prominent nucleoli. For HA, the major criteria are loss of hepatic plates, large but uniform nuclei, rare mitosis. For HCC, the criteria are loss of hepatocyte cell plates, large and variable nuclear size, prominent nucleoli and the presence of multiple nucleoli. For HB, the cells are embryonal, small and more basophilic, have scant cytoplasm and large nuclei with prominent nucleoli.

Following deparaffinization and hydration with xylene and graded alcohols, formalin-fixed, paraffin embedded slides were mordanted in 3% Acetic Acid (Rowley Biochemical Inc., E-323-3) for three minutes then stained with Alcian Blue Solution (Rowley Biochemical Inc., E-323-1) for 30 min (pH 2.5). Slides were then washed for 10 min in running tap water, followed by a deionized Water rinse. Slides were counterstained in Nuclear Fast Red Solution (Rowley Biochemical Inc., E-323-2) then dehydrated and cleared through graded alcohols and xylene and coverslipped with Micromount (Leica cat# 3801731, Buffalo Grove, IL) using a Leica CV5030 automatic coverslipper.