Immpress excel amplified hrp peroxidase polymer staining kit

Surgical resection procedures of GBM patients obtained tumor samples at the Departamento de Neurocirugía, Instituto de Neurocirugía Asenjo, Santiago, Chile. All procedures were carried out with the approval of the Bioethics Committee of the Universidad Austral de Chile (Permit Number: 29–2011) and Servicio de Salud Metropolitano Oriente. Human tissues were fixed in 3.7% paraformaldehyde (Sigma Aldrich, Darmstadt, Germany) and embedded in paraffin. Then, 5 μm sections were mounted on silanized slides and immunodetections were performed with primary anti-MRP3 (NBP1-42347) from NOVUS and anti-A2BAR (ab40002) from ABCAM in blocking solution overnight at 4 °C. Immunosignals were visualized using the ImmPRESS™ Excel Amplified HRP (peroxidase) Polymer Staining Kit (Vector Laboratories, Newark, CA, USA). Counterstaining was performed using hematoxylin and eosin classic stain.

The anti-cytokeratin 8 monoclonal antibody (Cytokeratin CAM 5.2 BDBiosciences-345779) was used to label MCF10A cell invasion into the chicken membrane. Slides were deparaffinised, blocked and incubated with anti-Cyt8 antibody (1:2 dilution), HRP-conjugated secondary antibody followed by 3,3′-diaminobenzidine (DAB) staining using the ImmPRESS™ Excel Amplified HRP peroxidase Polymer Staining Kit (Vector Laboratories MP-7602-15), according to the manufacturer's protocol. All images were captured as Z-stacks of brightfield microscopy with a 40× objective on the Olympus VS120 Virtual Slide Scanner Microscope.