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.
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.
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