Dako envision flex high ph detection kit

IHC staining was performed on the Dako Autostainer Plus using the Dako EnVision^™ FLEX High pH Detection Kit (K8010) (Dako, Carpinteria, CA). Slides were deparaffinized to DI water. Antigen Retrieval was performed on Dako PT Link water bath. The antigen retrieval was done at 97°C for 20 minutes. The slides were cooled until they reached 65°C. All slides for all antibodies were placed in Tris/EDTA pH 9 (Dako TRS High pH). Slides were washed in Dako wash buffer for 5 minutes. Slides were subjected to a peroxidase Block for 5 minutes. Slides were rinsed twice with wash buffer. Slides were incubated with primary antibody CLPTM1L (rabbit polyclonal, Sigma Aldrich cat# HPA014791, lot A57952) diluted to 1:400 for 30 minutes. Slides were rinsed with wash buffer. Slides were incubated with secondary antibody for 20 minutes and rinsed twice with wash buffer. Slides were incubated with DAB substrate for 10 minutes and rinsed with wash buffer. Slides were stained with hematoxylin for 7 minutes and rinsed with DI water. Slides were dehydrated and coverslipped for viewing. Staining intensity in tumor tissue was scored by three pathologists and averaged. Tumors were scored as negative – 0, weak – 1, intermediate – 2, or strong – 3. Independent scores were averaged. Antibodies for IHC on xenograft tumors were Ki-67 (CRM 325 A,B,C) and Cleaved Caspase-3 (Asp175, Cell Signaling, Boston MA) 1:300.

TfR1, H-ferritin, L-ferritin, and RRM2 expression in normal brain and glioblastoma, and TfR1 expression in human brain microvascular endothelial cells were examined by immunohistochemical (IHC) staining of tissue samples from surgically resected glioblastoma tumors from patients or from animal experiments. Dead cells in tumor xenografts in GaM-treated animals were identified according to typical nuclear morphological changes: pyknosis (nuclear condensation), karyorhexis (nuclear fragmentation), and karyolysis (complete dissolution of the nuclear fragments). Percent dead cells were calculated out of a total of 1000 cells (viable and dead) counted. Human tissue was obtained from the Brain and Spinal Cord Tissue Bank of the Medical College of Wisconsin. IHC staining with specific primary antibodies was performed on a Dako Autostainer Plus Instrument using the Dako EnVision™ FLEX High pH Detection Kit protocol. Stained slides were visualized using a Nikon Eclipse 80i microscope equipped with a MicroPublisher 3.3 RTV color video camera (Q Imaging, Surrey, BC, Canada). The images were captured using NIS elements imaging software (Version 7.0, Nikon Instruments, Inc.).