Il 6r alpha antibody

Two human ovarian cancer cell lines, Hey and SKOV3; and patient-derived xenograft (PDX) cell line (MDACC-HGSC-1, derived from human HGSC) were cultured in a corresponding medium as described previously [6 (link), 7 (link)]. The origin and purity of all cell lines were verified with short tandem repeat (STR) analysis before their use in MD Anderson’s Characterized Cell Line Core (https://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/characterized-cell-line-core-facility/index.html).
Paclitaxel (500 nM, Sigma), recombinant human IL-6 protein (R&D Systems, Minneapolis, MN, USA), IL-6R alpha antibody (R&D Systems, for in vitro use only), IL-6R alpha antibody (tocilizumab, for in vivo use in PDX models; Genentech, San Francisco, CA, USA), and apigenin (Sigma) were used in test groups as described in detail below.

Neutrophils and PBMCs were fixed in 3.5% paraformaldehyde before three washes in PBS. Cells were cytocentrifuged onto polylysine slides, permeabilised with Triton-X-100 (0.5%) for 10 min and rinsed in PBS. Cells were blocked with 0.5% BSA for 30 min before incubation with antibody sIL-6R AF or isotype control and staining with DAPI based mountant. Immunofluorescence was visualized using a fluorescent microscope.
For immunocytochemistry pelleted neutrophils and PBMCs were placed into induced plasma/thrombin clots, formalin-fixed overnight and processed into paraffin wax block. 4µm sections were placed onto adhesive slides and high pH antigen retrieval performed using a Dako PT Module. Slides were washed in PBS-Tween, blocked with Hydrogen Peroxidase solution (Dako, UK) and stained with IL-6R alpha antibody (R&D Systems) or mouse IgG₁ isotype (R&D Systems) control for 1 h at room temperature. Slides were washed as before, incubated with ENVision Flex HRP (Dako, UK) for 30 min and visualized with 3,3’-Diaminobenzedine (Dako, UK) to create a brown reaction product, counterstained with haematoxylin and examined by light microscopy.