Dako autostainer 48

For RANK immunohistochemical analysis, formalin-fixed paraffin embedded sections from BIA-ALCL patients were dewaxed in xylene prior to rehydration using graded ethanol concentrations. Antigen retrieval and immunostaining were performed as described [51 (link)] using monoclonal mouse anti-human RANK antibody (N-1H8; Amgen Inc.). For PDGFRA immunohistochemical analysis, monoclonal mouse anti-PDGFR-α (C-9) (Santa Cruz, sc-398206, dilution 1:100) was incubated overnight at 4°C following antigen retrieval with Dako Low pH 3 in 1 buffer at 97°C for 20 minutes in a Dako PT Link (Agilent Technologies, K8005). Visualization was performed on a Dako Autostainer48 using Envison Flex detection kit (Agilent Technologies, K5007).

To characterise the xenograft models, immunohistochemical analysis of SSTR2 expression on tumour tissue was performed. Untreated xenografts of IMR-32 (N = 6) , Neuro 2a (N = 3) and SK-N-AS (N = 2) were used. Furthermore, histological analysis of kidneys from animals taken 24 h (N = 7, n ≥ 2) or 6–11 weeks (N = 15, n ≥ 3) after last treatment was performed to assess toxicities related to the mono- and combination treatments. For more details on the treatments, see Therapy study section below.
All tissues were fixed in 4% buffered formalin, paraffin-embedded, sectioned and deparaffinised. Antigen retrieval was performed using low pH retrieval solution (Dako K8005, Agilent). Staining was performed using EnVision Flex Kit with DAB as chromogen and a Dako Autostainer 48 (Agilent, USA). Sections were immunostained with an antibody against SSTR2 (ab134152, Abcam, diluted 1/1000) and detected with EnVision Flex Kit (Dako K8010, Agilent). Counterstaining with haematoxylin (Histolab, Sweden) was performed in a Tissue-Tek Prisma (Sakura, Netherlands).

The list of antibodies and relevant details of immunohistochemical protocols are summarized in Table 1. Heat induced epitope retrieval (HIER) was used for antigen retrieval, employing different pH according to the antibody. For EGFR, a proteolytic pretreatment step was used. Section staining was carried out on Benchmark Ultra stainer (Ventana/Roche, Tucson, AZ, USA) using either the Ventana ultraView Universal DAB detection kit or the Ventana OptiView DAB IHC detection kit; both methods use the avidin-biotin complex method with horseradish peroxidase as an enzyme and DAB (3,3′-diaminobenzidine) as a chromogen. An Agilent/Dako Autostainer 48 (Agilent, Santa Clara, CA, USA) using the PT-Link pretreatment system and the EnVision Flex detection kit with DAB was used for INSM1. Subsequently, all slides were counterstained with haematoxylin. Normal placental tissue was used to set up EGFR assay. Positive control on slide was used with different tissues according to the antigen detected antigen (Figure 1). For EGFR amplification detection in 4 TMAs with GBM, silver ISH with EGFR DNA Probe (Ventana/Roche) and ultraView SISH Detection Kit (Ventana/Roche) was employed on a Benchmark Ultra stainer. Additional GBMs cases without enough representative material in TMA were evaluated as whole sections using the ZytoLight SPEC EGFR/CEN 7 Dual Color Probe set (ZytoVision, Bremehaven, Germany).