Tissue tek coverslipping film

Tissue sections of 4 μm were deparaffinized, stained for 10 min in Mayers HTX
(Bio Optica/Dalab, Milano, Italy), rinsed, and stained for 1.5 min in eosin
(Histolab AB, Gothenburg, Sweden). The slides were then dehydrated, cleared, and
mounted using Tissue-Tek coverslipping film (Sakura Finetek, Torrance, CA). An
experienced pathologist blinded to the clinical data marked the tumor area in
tissue sections from all biopsies and determined the Gleason score of the
untreated tissue. The tissue was graded as follows: 3 + 3 (IUSP grade 1), 3 + 4
(ISUP grade 2), 4 + 3 (ISUP grade 3), 4 + 4 (ISUP grade 4), and 4 + 5 or higher
(ISUP grade 5).

Sections of 4 μm tissue taken serially after the mIHC stained section were
deparaffinized followed by antigen retrieval in EnVision FLEX Target Retrieval
Solution, high pH (Dako, Glostrup, Denmark) using PT-link at 97°C for 20 min.
Slides were then incubated for 30 min at room temperature with the monoclonal
mouse anti-human-CD163 antibody (clone 10D6, 1:200, Novocastra, Leica
Microsystems, Newcastle, UK). The immunohistochemical EnVision visualization
system was used with the standard method of horseradish peroxidase and
3,3’-diaminobenzidine, incubating the sections with a dextran polymer conjugated
with secondary antibodies for 20 min and substrate working solution FLEX DAB
sub-chromophore for 5 min in Autostainer Link 48 according to the manufacturer’s
instructions (Dako). Counterstaining was performed using Mayer’s hematoxylin,
and slides were dehydrated, cleared, and mounted using Tissue-Tek coverslipping
film (Sakura Finetek). Tonsil tissue was used as a positive control for the
CD163 antibodies. A person with experience in assessing CD163 stained cells
inspected the whole biopsy and graded the tissue as (a) 20% or less of the
stroma cells expressing CD163 and (b) more than 20% of the stroma cells
expressing CD163. In cases where there were grading difficulties, an experienced
pathologist was consulted.

For viral antigen staining, a Roche Optiview DAB IHC kit, in combination with an anti-SARS-CoV-2-nucleoprotein (clone E16C; ThermoFisher), was used in a Ventana Benchmark Ultra immunostainer (Roche, Basel Switzerland), as previously described [7 (link)]. In brief, antigen retrieval took place with cell conditioning 1 (CC1, Ventana Medical Systems) (pH 8.5) for 24 min at 100 °C, 1/5.000 diluted. Thereafter, incubation took place with the primary antibody for 48 min at 36 °C, followed by standard Optiview detection/visualization with DAB and Copper.
After immunohistochemical staining, the sections were dehydrated and mounted with TissueTek^® coverslipping film (Sakura Finetek Europe B.V., The Netherlands). Hematoxylin-eosin (HE) staining was used for general morphology. Cells were counted in a blind manner. As a positive control, a lung section of a deceased COVID-patient was included (Figure S1).