Zytochem plus hrp polymer kit

Paraffin sections were incubated at 60 °C for 2 h and afterwards dehydrated with xylene and decreasing ethanol concentration, followed by distilled water. Antigen retrieval was performed using 10 mM sodium citrate solution (pH6) for 30 min in a water bath at 95 °C. The endogenous peroxidase activity of the sections was blocked by using 3% H₂O₂ for 10 min. Antigen detection, including blocking of background staining, was performed using a ZytoChem Plus HRP Polymer Kit (Zytomed Systems GmbH, Berlin, Germany). Tissue sections were incubated with primary antibodies for 30 min at 37 °C (Ki67 monoclonal antibody 1:10 (SP6, Thermo Fisher Scientific, Rockford, IL, USA ), MelanA monoclonal antibody 1:50 (A103, Abcam, Cambridge, UK), and antihuman melanosome 1:100 (HMB45, Dako, Carpinteria, CA, USA), followed by DAB/HRP detection. Sections were counterstained with Mayer’s hematoxylin solution for 30 s.

The tissue samples were immediately processed further for cryopreservation. They were flash-frozen (Tissue-Tek, O.C.T.) and cut longitudinally (7 μm width) using a cryostat. Giemsa staining and immunostaining (ZytoCHEM-Plus HRP Polymer-Kit, ZYTOMED Systems) were performed after formalin fixation with antibodies against alpha-smooth muscle actin (αSMA) as a myofibroblast marker (Abcam; ab7817; 1:100), cluster of differentiation 68 (CD68) as a marker for macrophages (DakoCytomation; M0718; 1:50), collagen type I (Abcam; ab6308; 1:100) and collagen type III (Abcam; ab7778; 1:500).

A total of 7 TMA blocks containing 199 consecutive cases were constructed by inserting cylindric tissue cores measuring 2 mm in diameter into a paraffin block. For each tumor and non-malignant tissue 2 cores were embedded. Sections of each TMA block were mounted on silane-coated slides and subsequently further processed for immunohistochemistry as described before [29 (link)]. In short, antigen retrieval was performed by 5 min cooking in citric buffer (pH = 6.0). For blocking ZytoChem Plus (HRP) Polymer Kit (Zytomed, Berlin, Germany) was used according to manufacturer’s instructions. Primary antibodies against CCL22 (Peprotech, Hamburg, Germany), FoxP3 (Abcam, Cambridge, USA) and CCL1 (Atlas antibodies, Stockholm, Sweden) were incubated for 16 h at 4 °C. Subsequent to 30 min of incubation with a horseradish peroxidase-polymer (Zytomed, Berlin, Germany) staining was carried out using 3,3-diaminobenzidine-substrate solution (DAB) (Dako, Glostrup, Denmark).

Construction of tissue microarray (TMA) blocks including all cHL cases has been described previously [3 (link)]. For each patient, two 1-mm-diameter cores, selected from two different representative tumor areas rich in Hodgkin and Reed-Sternberg cells, were included. All cases showed cores with representative tumor microenvironment and high numbers of HRS cells (from 10 to 178 neoplastic cells/mm², median: 70 cells/mm²). Buffers used for antigen retrieval and primary antibodies are listed in (Table A in S1 File). The double immunohistochemistry methodology has been described previously [28 (link)]. pSTAT1 (polyclonal, Santa Cruz Biotechnology, Dallas, USA) or CMAF (M-153, Santa Cruz Biotechnology, Dallas, USA) antibodies were used as first primary antibodies and the detection of bound antibodies was performed using ZytoChem Plus HRP polymer kit (Zytomed Systems, Berlin, Germany), employing diaminobenzidine (DAB) as chromogen. CD68 (clone PGM1, Dako, Gloustrup, Denmark) or CD163 (clone 10D6, Novocastra, Wetzlar, Germany) antibodies were incubated posteriorly, followed by detection with AP Polymer System (Zytomed Systems, Berlin, Germany), employing Blue Alkaline Phosphatase substrate kit (Vector Laboratories, Burlingame, USA) as substrate. The sections were not counterstained.

Immunohistochemical staining was performed on paraffin sections (2 μm thickness). All sections were deparaffinized with xylene twice, rehydrated using decreasing ethanol concentrations, and then subjected to heat-induced epitope retrieval in antibody buffer. Stainings were performed using the antibodies listed in Supplementary Table S1, the ZytoChem Plus HRP Polymer Kit (Zytomed Systems, Berlin, Germany), 3,3′-diaminobenzidine solution (DAB), hematoxylin counterstain and the Eukitt mounting medium. Stained slides were digitized using the Nano Zoomer S210 (Hamamatsu Photonics, Herrsching a. Ammersee, Germany). DAB-positive and negative cells were counted with the nuclei detection image analysis application of Visiopharm (Visiopharm, Hørsholm, Denmark).

Samples were fixed in 4% formaldehyde, followed by dehydration, paraffin embedding, sectioning (2.5 µm), and standard hematoxylin and eosin (H&E) staining. Immunohistochemistry (IHC) was performed using the ZytoChem Plus HRP Polymer Kit (Zytomed Systems GmbH, Bargteheide, Germany, POLHRP-100) and DAB Substrate Kit (Zytomed Systems GmbH, DAB057) according to the manufacturer’s protocol. IHC was performed using antibodies listed in Table S4. Images were captured with ScanScope (Leica, Wetzlar, Germany) or EVOS M7000 microscope (Thermo Fisher) and processed with Aperio ImageScope (version 12.4.6.5003), Image J (version 1.53e), and Photoshop (version 13.0.1).

For immunohistochemistry, heat‐induced epitope retrieval was performed either with citrate or with EDTA buffer according to the manufacturer’s protocol of the respective primary antibody. Staining was performed on a Leica BOND‐MAX. Sections were incubated for 1 h with the following primary antibodies: mouse monoclonal anti‐calretinin antibody (Dako M 7245, clone DAK Calret 1; 1 : 100), mouse monoclonal anti‐EPCAM antibody (Ber‐EP4; Dako M 0804, Clone Ber‐EP4; 1 : 50), and mouse monoclonal SMA antibody (Progen Biotechnik GmbH, Clone ASM‐1; 1 : 200). Sections were incubated with the primary antibody for 20 min, except for SMA (30 min). Specimens were washed and incubated with postblock solution and HRP‐polymer reagent according to the manufacturer’s protocol (ChromoPlex 1 Dual Detection Kit for BOND; Leica). For TWIST and p53, staining was performed on a DAKO Autostainer Plus. Sections were incubated for one hour with mouse monoclonal anti‐TWIST antibody (1 : 200; Abcam ab175430, Berlin, Germany, Clone 10E4e6) or mouse monoclonal p53 antibody (1 : 50; Dako M 7001, Clone DO‐7). Sections were washed and incubated with postblock solution and HRP‐polymer reagent according to the manufacturer’s protocol of ZytoChem Plus HRP Polymer Kit (Zytomed Systems, Bargteheide, Germany).