Ventana benchmark xt processor

Tissue sections were stained with Estrogen Receptor (SP1, CONFIRM), Progesterone Receptor (IE2, CONFIRM), and HER2 (4B5, PATHWAY). Immunohistochemistry was performed using the UltraView Universal DAB Detection Kit (Ventana). The immunohistochemistry of NIK (1:500, ab220442) and ALDH (1:500, 611195, BD) was performed using a Ventana Ultraview DAB detection kit (Ventana) and UltraView Universal Alkaline Phosphatase Red Detection Kit (Ventana), respectively. Diaminobenzidine or Fast Red was employed as the chromogen, and hematoxylin as the nuclear counterstain. All slides were processed in a Ventana BenchMark XT processor (Ventana, Tucson, AZ, USA). Slides were scanned in ScanScope CS2 (Aperio) and image analysis was conducted in ImageJ. This study has used paraffin embedded breast cancer tissue obtained from the Oncology Hospital in the XXI Century National Medical Center. All used tissues were part of residual material and they did not endanger patients diagnostic.

Samples of skin, lung, liver, spleen and small/large intestine were examined on day 56 or at the time of death post transplantation. These samples were fixed with 10% formalin, embedded in paraffin, and stained with Hematoxylin and Eosin (H&E) and Masson’s trichrome. Disease was quantified blindly by one of the investigators (SN) using a modified cGvHD pathology scale as previously described[17 (link)]. Twenty parameters for lung, seventeen for liver and two for spleen were scored from 0 to 4 (S1 Table). Trichrome-stained slides were digitized with a ScanScope (Aperio, Vista, CA). Collagen depositions were quantified on at least 3 different randomly selected sections in the alveolar area of the lung and the whole liver (magnification 20x) as a ratio of blue staining area to the total staining area using Aperio ImageScope. For some experiments, blocks were stained with anti-human CD4 (Ventana prediluted), anti-human CD8 (DAKO, 1:20), anti-human CD20 (Ventana prediluted), anti-human CD68 (Ventana prediluted) and anti-human FoxP3 (eBioscience 1:20) using a Ventana Ultraview DAB detection kit in a Ventana BenchMark XT processor (Ventana, Tucson, AZ).

Formalin-fixed paraffin-embedded (FFPE) blocks with ESCC and adjacent non-tumor tissue samples were obtained from the Department of Pathology in our hospital. Briefly, FFPE tissue sections (4 μm) were dewaxed and rehydrated. After antigen retrieval at 37°C for 2 hours in a Ventana BenchMark XT processor (Ventana, Tucson, AZ), tissue sections on the slides were incubated with primary antibodies. After reincubation with polymer-horseradish peroxidase reagent, the sections were visualized with Ventana ultraview DAB (diaminobenzidine 3,3′ tetrahydrochloride) detection kit according to the manufacturer's protocol. The primary antibodies used were a rabbit polyclonal antibody against AXL (1:100, ab72069, abcam) and HER2 (ready- to-use, DAKO). The tissue slides were then counterstained with hematoxylin.