Medical decloaking chamber

Archival formalin fixed paraffin embedded (FFPE) tissue microarrays were sectioned at 3µm on superfrost+ slides. Sections were dewaxed in xylene, rehydrated in descending concentrations of ethanol to water and endogenous peroxidase quenched with 2% H₂O₂ in pH 7.5 Tris-buffer saline for 10 min. Antigen retrieval was carried out in a Biocare Medical Decloaking Chamber using Dako Target Retrieval Solution pH 9 for 15 min at 105°C. To eliminate nonspecific background staining, sections were blocked with 3% hydrogen peroxide in tris-buffered saline with 0.01% Tween-20 (TBS-T) for 5 minutes and Biocare Medical Background Sniper in 2% BSA for 15 min.
For chromogenic staining, Sigma Rabbit anti-human C9 primary antibody (Sigma Aldrich #SAB4503059) was diluted 1:300 in Background Sniper + 2% BSA for 60 minutes at room temperature. The specificity of this C9 antibody in IHC was previously assessed (7 (link)) by incubation of the antibody with recombinant purified C9 prior to using the antibody for IHC. Staining was visualized using Biocare Medical MACH2 Rabbit Polymer HRP detection system applied for 30 minutes, and Betazoid DAB for 5 minutes. Sections were washed, and counterstained in Mayers’ haemotoxylin, dehydrated through ascending graded alcohols, cleared in xylene, and mounted using DePeX. IHC images were obtained in Aperio AT turbo digital slide scanner from Leica Biosystems.

Human primary and matched recurrent GBM tissues were obtained from the Northwestern University’s Nervous System Tumor Bank. All patients were consented according to the Institutional Review Board (IRB) policies prior to the obtainment of samples. Samples were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemistry of tumor samples was performed on 4-μm-thick sections heated at 60 °C for at least 1 h. Staining for IL-8 was carried out manually, and antigen retrieval was performed with a BioCare Medical Decloaking Chamber using high (LC3) or low pH antigen retrieval buffer from Dako. Primary antibodies were incubated for 1 h at room temperature. A secondary antibody was EnVision-labeled polymer-HRP (horseradish peroxidase) anti-mouse or anti-rabbit as appropriate. Staining was visualized using 3, 3′-diaminobenzidine (DAB) chromogen (Dako, K8000).
IL-8 immunohistochemical results on TMAs were semiquantified on a relative scale from 0 to 3, with 0 = negative and 3 = strongest (see Supplementary Fig. 1). Each tumor was represented by three separate cores on three separate blocks.