Cytoseal 60

For comparative purposes, the mappings sometimes included immunochemical detection of calbindin (CB), calretinin (CR), distalless homeobox protein (DLX), nitric oxide synthase (NOS), neuropeptide Y (NPY), parvalbumin (PV), paired box protein 6 (PAX6), T‐box, brain, 1 protein (TBR1), and tyrosine hydroxylase (TH).
Our immunohistochemical reaction protocol was described in detail elsewhere (Ferran, Ayad, et al., 2015). The primary antibodies used are described in Table 3. After washes, the sections were incubated with biotinylated goat anti‐rabbit or goat anti‐mouse (Vector Laboratories, CA; used at 1:200 dilution) followed by a streptavidin–peroxidase complex (Vectastain‐ABC kit; Vector Laboratories; 0.001% dilution), applied for 1 hr at room temperature. Peroxidase activity was developed with 0.03% 3,3′‐diaminobenzidine (Sigma; St Louis, MO), plus 0.003% hydrogen peroxidase. After immunohistochemical and hybridization labeling, the slides were washed several times in PBS, air‐dried and coverslipped with Cytoseal 60 (Thermo Scientific, Ref. 8310‐16) or Mowiol (Calbiochem, Bad Soden, Germany, Ref. 475904). We verified the specificity of the antibodies by performing parallel control experiments that omitted the primary antibody, checking that no residual immunostaining was detected (data not shown).

Immunohistochemical analysis of tumour samples stored as paraffin-embedded tissue was carried as previously described [42 (link)]. Laminin (1/100) and cleaved caspase 3 (1/100, #9664S, Cell Signaling, MA, USA) antibodies were used. Depending on the technique performed and the manufacturer’s specifications, different antibodies were used to detect myoepithelial cells: CD10 (1/100, #M7308, Dako, CA, USA), p63 (1/100, #sc-8431, Santa Cruz Biotechnologies, TE, USA) and αSMA (1/100) were used as primary antibodies. After washing the samples were incubated in the presence of HRP-conjugated secondary antibodies. After washing, samples were incubated with Vectastain ABC for 30 min in a humidified chamber followed by incubation in the presence of DAB substrate (FAST™ 3,3′-diaminobenzidine tablets, Sigma) for 5–10 min at RT, monitoring colour development by microscopy. Slides were washed with water and counterstained in 1:3 Gill II haematoxylin (Panreac) for 1 min. The slides were mounted with Cytoseal™ 60 (Thermo Scientific), left to dry and analysed by phase contrast microscopy.

Slides were incubated with SYNGR3 Invitrogen antibody at 1:300 for 1 hour and were detected with ImmPress HRP anti-rabbit IgG and TSA Cy5 (SAT705A001EA). After completion of SYNGR3 staining, a second round of denaturation (10 minutes, Bond-Epitope Retrieval solution 1) was followed by incubation in either anti- pan-Cytokeratin (30 minutes, 1:1,500) or CD45 (30 minutes; ready to use) and detection with ImmPress HRP anti-rabbit IgG and TSA Cy3 (SAT704A001EA; Perkin Elmer). Following pan-Cytokeratin/CD45 staining, a third denaturation step was performed for 10 minutes in Bond-Epitope Retrieval solution 2 (pH 9.0; AR9640) followed by incubation with either anti-CD3 (1 hour, 1:200) or p16 (1 hour, 1:5) then detection with Bond Polymer (DS9455) and TSA Alexa-488 (B40953, Invitrogen).
Stained slides were dehydrated and coverslipped with either Cytoseal 60 (single DAB stains; 8310-4, Thermo Fisher Scientific) or Prolong gold (multiplex stains; P36930, Thermo Fisher Scientific). Positive and negative controls (no primary antibody) were included during staining runs. The slides were digitally scanned at 20× magnification using Aperio AT2 (Aperio Technologies) and uploaded to the Aperio eSlideManager database (Leica Biosystems Inc) at the Pathology Services Core at UNC.

FFPE bone tissue with PCa metastases was subjected to RNA in situ hybridization (RISH). The RNAscope^® Intro Pack 2.5 HD Reagent Kit Brown-Hs (ACDbio, CA; 322370) was used following the manufacturer’s instructions exactly. The HybEZ^™II system was used for all incubation steps warmer than room temperature. The hybridization probes were designed and purchased from ACDbio: SULF1-Hs (403581), HSPG2-Hs (573501), Wnt3A-Hs (429431). Negative, DapB, and positive, PPIB, control probes were used in every experiment. After the signal development step (chromogenic, DAB), each section was counterstained for exactly 10 seconds with hematoxylin QS (Vector Laboratories, CA; H-3404), and lastly, mounted in Cytoseal^™60 (Thermo; 8310-16). After curing of the mountant, the entire section for each sample was imaged via color brightfield, 40X objective, using the Keyence automated microscope BZ-X8100 (Keyence, Japan).