Dako envision system hrp labeled polymer

After completion of mouse treatments, the xenograft tumors were isolated, fixed in formalin, and paraffin‐embedded. Tissue sections (4 μm) were deparaffinized in xylene and rehydrated via graded ethanol. The sections were subjected to heat‐induced antigen retrieval in sodium citrate buffer (10 mm, pH 6) at 104 °C for 20 min, and treated with H₂O₂ (0.02%) for 10 min to inactivate endogenous peroxidases. The sections were blocked with Dako Ready‐to‐use Protein Block Serum‐Free (X0909; Dako North America, Inc., Carpinteria, CA, USA) for 15 min, and then incubated overnight with p‐AXL (Y799; 1 : 200 dilution), Ki‐67 (1 : 200 dilution), or cleaved caspase‐3 (1 : 400 dilution) primary antibodies. Next, the sections were incubated with Dako EnVision+ System‐HRP labeled Polymer (K4002; Dako North America, Inc.) for 30 min, followed by the application of 3, 3′‐diaminobenzidine (DAB) for 5 min, and counterstaining of the tissues with hematoxylin. Images were acquired by using an Olympus BX51 microscope (Olympus Co., Center Valley, PA, USA). The protein expression level of p‐AXL (Y779) was determined by using the IHC toolbox plugin in imagej software (https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html). Expression levels of Ki‐67 or cleaved caspase‐3 were reported as % of positive cells relative to total cell number in xenografts from four groups of mice.

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).