Three micrometer-thick sections immediately adjacent to sections cut for gene expression profiling were used to digitally quantify the inflammatory cell load in cases that had enough tissue in the FFPE tissue block after cutting the RNA curls (n = 83). In brief, after deparaffinization and low pH antigen retrieval, sections were incubated with anti- CD45 antibody (1:2000, clone: PD7/26 + 2B11, Dako-Agilent) followed by antigen visualization with Bond Polymer Refine Detection Kit (Leica Biosystems). Whole-slide digital images (WSDI) (Aperio ScanScope XT, Leica Biosystems) of the CD45-stained slides were analyzed by the Cell Count module of the Definiens Tissue Studio software. Inflammatory cell count (ICC) was defined by the average number of CD45-positive cells per 1 mm2 tissue area. For each WSDI, two inflammatory cell count metrics were generated: whole section ICC and cortical ICC. Cortical ICC measured the inflammatory cell density only in the cortex while perivascular lymphoid aggregates, large vessel lumina, the renal capsule, and non-renal tissue were excluded from the analysis.
Anti cd45 antibody
Manufactured by Agilent Technologies
Sourced in United States
The Anti-CD45 antibody is a laboratory reagent used for the identification and isolation of CD45-positive cells, such as leukocytes, in various research applications. It binds specifically to the CD45 protein expressed on the surface of these cells.
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2 protocols using anti cd45 antibody
1
Quantifying Inflammatory Cell Load in FFPE Tissue
2
Evaluating Oxidative Stress in ICU Patients
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Clinical variables such as serum lactate (mmol/L) and peripheral blood C-reactive protein (mg/L) were measured within 24 h of admission. To assess ROS production, a 10 mL aliquot of blood was drawn from the central line catheters as soon as possible once consent was obtained and no later than 24 h after admission to the ICU. The ROS production analysis was carried out on fresh samples for immediate collection. ROS production was measured in isolated lymphocytes with Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA) and they were labeled with anti-CD45 antibody (Agilent, Santa Clara, CA, USA). Subsequently, flow cytometry and dihydroethidium staining (DHE, Abcam, Cambridge, UK) were used to assess the production of ROS in the lymphocytes of each patient. Cells from both populations were stained with 2.5 µM DHE for 30 min, washed with PBS, and acquired on the Guava®-EasyCyte® 6-2L capillary cytometer (Luminex Corporation, Austin, TX, USA). Analysis of the mean fluorescence intensity for DHE was reported.
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