Rnascope 2.0 hd reagent kit

In situ hybridization was performed using the RNAscope 2.0 HD Reagent kit (Brown) (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions. Mouse AhR mRNA RNAscope probes were custom-made by Advanced Cell Diagnostics and targeted bases 867–1836 of mouse AhR mRNA (NCBI reference sequence: NM_013464.4). Probe sets specific for the housekeeping gene peptidylprolyl isomerase B (Ppib) from mouse and the dapB gene from Bacillus subtilis were used as positive and negative controls, respectively. After deparaffinization and dehydration, formalin-fixed sections were pretreated with protease and then subjected to in situ hybridization. Briefly, sections were hybridized with the probe solution, followed by amplification and probe detection using staining solutions. The sections were then stained with Gill’s hematoxylin to visualize individual cells in each brain region. Slides were covered with Marinol (Muto Chemicals, Tokyo, Japan) and a plastic coverslip before viewing with a Leica DM6000 B microscope (Leica Microsystems, Tokyo, Japan). Bright-field images were captured using a Leica DM6000 B microscope with specific objective lenses (HCX PL APO, 40 ×, NA = 0.75; Leica Microsystems).

In situ detection of PD-L1 transcripts in FFPE TMA samples was performed using the RNAscope® assay with Probe-Hs-PDL1-v2 (Cat# 409671, Advanced Cell Diagnostics, USA) and RNAscope® 2.0 HD Reagent Kit (BROWN) (Cat# 310035, Advanced Cell Diagnostics, USA) following the protocols suggested by the manufacturer. See Supplemental Materials and Methods for details on staining and scoring with Aperio Imagescope, Spotstudio and Cell profiler.

Cytokine mRNA production of IL6 and IL10 was further investigated by RNA in situ hybridization (ISH) using RNAscope technology. The RNAscope assay was applied to cell block paraffin sections of late seromas as previously described [16 (link), 17 (link)]. Briefly, FFPE Sects. 2 μm thick were deparaffinized in xylene and then hydrated in an ethanol series. Hybridization was with target probes: Probe-Hs-IL6 and Probe-Hs-IL10. The preamplifier, amplifier, label probe, and chromogenic detection procedures were performed according to the manufacturer’s instructions (RNAscope® 2.0 HD Reagent Kit, Advanced Cell Diagnostics, Newark CA, USA).
For double staining, RNAscope assay for IL6 and IL10 was performed first and followed by immunohistochemistry for CD30 (clone Ber-H2, diluition 1:50) (Dako, Denmark). Staining was revealed using Super Sensitive Link Label IHC Detection System Alkaline Phosphatase (BioGenex, Fremont, CA, USA). Vulcan Fast Red Chromogen Kit 2 (BioCare Medical, Pacheco, CA, USA) was used as substrate-chromogens, followed by counterstaining with Harris hematoxylin.

The RNAscope assay was applied to lymph node paraffin sections from three HL patients using probes to IL-31 and TSLP, as previously described [56 (link)]. Briefly, formalin fixed, paraffin embedded (FFPE) tissue sections 2 μm thick were deparaffinized in xylene and then hydrated in an ethanol series. Hybridization was performed with the negative control probe dapB, the positive control probe Probe-Hs-Ubiquitin, and the target probes Probe-Hs-IL-31 and Probe-Hs-TSLP. The preamplifier, amplifier, label probe, and chromogenic detection procedures were performed according to the manufacturer’s instructions (RNAscope^® 2.0 HD Reagent Kit, Advanced Cell Diagnostics, Hayward, CA, USA).

Two samples [1 histologic (Patient 1) and 1 cytologic (Patient 6)] were adequate to perform RNAscope ISH analysis. FFPE tissue and cell block sections 5 μm thick were deparaffinized in xylene and then dehydrated in an ethanol series. Hybridization was with target probes (probe symbols: Hs-Probe-Hs-ALK-E1-E18 and Hs-ALK-E19-E29-20P; probe name: anaplastic lymphoma receptor tyrosine kinase). The preamplifier, amplifier, label probe, and chromogenic detection procedures were according to the manufacturer's instructions (RNAscope^® 2.0 HD Reagent Kit, Advanced Cell Diagnostics, Hayward, CA, USA). The scoring guidelines and interpretation were in accordance with those reported by Wang et al. [26 (link)].