Immunohistochemical Analysis of PREX1 in Glioblastoma

A glioblastoma tissue microarray consisting of 35 cores of glioblastoma tissue, two cores of adjacent brain tissue and three cores of normal brain tissue, all in duplicate, was purchased from US Biomax (GL805a F113, Rockville, MD, USA). Immunohistochemistry was done as described previously [21 (link)]. Antigen retrieval was performed in citrate buffer, pH 6.0 (Vector, Burlingame, CA, USA) in a decloaking chamber using the instrument's default program (Biocare Medical, CA, USA). Slides were incubated with PREX1 [D8O8D] rabbit monoclonal at a concentration of 4 ug/ml at 4°C overnight. The DakoEnVision+ system HRP labeled polymer was used for detection of bound antibody (Dako North America, Carpinteria, CA, USA) and sections were developed using DAB Peroxidase Substrate Kit (Vector, Burlingame, CA, USA) and counterstained with haematoxylin (Vector; Burlingame, CA, USA). Slides were digitized using the ScanScope CS2 (Aperio, CA, USA). The tissue microarray was scored independently by MD and JW and discrepancies were averaged. Signal intensity per core was scored as 0, 1, 2, or 3. Frequency of positive staining per core was scored as 0 (0% of cancer cells positive), 1 (0% to 33% of cancer cells positive), 2 (33% to 66% of cancer cells positive), and 3 (66% to 100% of cancer cells positive).