Vulcan fast red chromogen kit 2

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.

Double IHC was also performed as previously described^{23 (link),24 (link)}. The conditions for sections, blocking, antigen retrieval and secondary antibodies were the same as those for single IHC. Positive reactions were detected as brown colouration with 3,3′-diaminobenzidine tetrahydrochloride, the Vulcan Fast Red Chromogen Kit 2 (BIOCARE MEDICAL, Concord, CA, USA) and the HistoGreen Substrate Kit for Peroxidase (Linaris, Heidelberg, Germany). Sections were then counterstained with haematoxylin.
The following three items (c, d) were examined: (c) The number of cells positive for each macrophage marker around the lymphatic vessels in the border area in PTC and FTC was counted in 5 fields obtained at a 40 × microscopic power. (d) The number of cells positive for M1 and M2 markers inside and outside the areas of lymphatic invasion in PTC was counted. In this study, CD68 (PG-M1 or KP-1)⁺CD163^-CD206^- cells were defined as M1, and CD163⁺CD206⁺ cells were defined as M2. Specimens in which TTF-1⁺ PTC cells were observed in D2-40⁺ lymphatic vessels were designated as containing areas of lymphatic invasion. Furthermore, the intraluminal area of the lymphatic vessel with lymphatic cancer invasion was considered inside the lymphatic wall, and the area surrounding the lymphatic vessel with lymphatic cancer invasion, 100 μm in width, was considered outside the lymphatic wall.

Immunohistochemistry was performed on 5-μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections. Sections were put in 0.2% potassium permanganate solution for 20 min and 1% oxalic acid solution in 10 s for depigmentation, following deparaffinization and antigen retrieval. Sections were incubated at 4°C overnight with EZH2 Rabbit Antibody (1:1000, Cell Signaling Technology, 3147, Beverly, MA, United States) and H3K27me3 Rabbit Antibody (1:1000, Cell Signaling Technology, Beverly, 9733, MA, United States). We used Vulcan Fast Red Chromogen Kit 2(Biocare Medical, 1-800-799-9499) as a chromogenic reagent. The slides were scanned automatically by a NanoZoomer Digital Pathology System. The acquired pictures were viewed with NDP view software. We counted 1,000 cells and recorded the percentage of positive cells. We discretized the continuous data of EZH2-positive cell percentage to the categorical data from 0 to 3 (0, no staining; 1, ≤10%; 2, ≤30%; 3, >50%). The results were then subdivided into low (score = 0, 1) and high (score = 2, 3) expression groups.

Immunostaining and Congo red histochemistry were performed on 5 μm paraffin sections. Antigens were retrieved in 25 mM citrate buffer (pH 7.2) or Rodent Decloaker (Biocare Medical, USA) using a microwave oven. Sections were blocked with M.O.M.™ IgG block (Vector Labs, USA) or Rodent Block M (Biocare Medical). Primary antibody (Additional file 1: Table 1) incubation was carried out overnight at ~ 4 °C followed by incubation with secondary antibody for 30–60 min at room temperature. ABC™ complex and NOVA™ red reagents (Vector Labs) were used to visualize the immunosignals. In other settings, MM AP-Polymer kit (mouse antibody on mouse tissues) or MACH3™ Rabbit-Probe Alk Phos Polymer kits were employed along with Vulcan Fast Red Chromogen kit 2 (Biocare Medical). For double immunostaining using fluorescent secondary antibodies, primary antibodies were incubated overnight, simultaneously or stepwise at 4 °C, followed by incubation with the appropriate secondary antibodies (Alexa Fluor, fluorescent secondary antibodies against mouse and rabbit, Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used for counterstaining of nuclei. Sulfated alcian blue staining was performed according to Lendrum et al. [28 (link)].

Formalin-fixed, paraffin-embedded skin sections were stained with hematoxylin and eosin (H&E) or with primary antibodies against BrdU and mouse KLK6 at 4°C overnight followed by anti-mouse horseradish peroxidase- and anti-rabbit alkaline phosphatase-linked secondary antibodies from MACH 2 Double Stain 2 (Biocare Medical, Concord, CA). Immunoperoxidase reaction was visualized using the Dako Envision system plus HRP/DAB Kit (Dako, Tokyo, Japan) and alkaline phosphatase activity was detected by Vulcan Fast Red Chromogen Kit 2 (Biocare Medical, Concord, CA).