Expression of arginase I and inducible nitric oxide synthase in the peripheral blood and lymph nodes of HIV-positive patients

ZHANG, NAICHUN; DENG, JIANNING; WU, FENGYAO; LU, XIANGCHAN; HUANG, LEI; ZHAO, MIN

Detection of the expression levels of Arg I and iNOS using streptomycin avidin-biotin-peroxidase complex immunohistochemical analysis. The LNs were collected, fixed in 4% paraformaldehyde (Beijing Chemical Works, Beijing, China), embedded in paraffin (Leica Biosystems, Nussloch, Germany) and sectioned (4 µm). Briefly, the slides were washed in xylene (ZSGB-BIO, Beijing, China) to remove the paraffin, rehydrated through serial dilutions of alcohol (Beijing Chemical Works), followed by washing with phosphate-buffered saline (PBS; pH 7.2; ZLI-9062, ZSGB-BIO). Treated slides were then placed in a 0.01-mol/l citrate buffer (pH 6.0; ZSGB-BIO) and heated in a microwave oven for 15 min for retrieval of antigens, treated with 1.5% H 2 O 2 (Beijing Chemical Works) in methanol (Beijing Chemical Works) to inactivate endogenous peroxidase and treated with fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) to block non-specific binding. The sections were then incubated overnight at 4˚C with anti-hArginase1 antibody [cat. no. IC8026P; monoclonal mouse immunoglobulin (Ig) G2B; clone 658922; 1:350 dilution; R&D Systems, Minneapolis, MN, USA], anti-iNOS antibody (cat. no. ab15323; rabbit polyclonal; 1:80 dilution; Abcam, Cambridge, UK) and washed three times with PBS, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. D39-110; Polink-2 Plus HRP Rabbit for DAB Bulk kit; or cat no. D37-110; Polink-2 Plus HRP DAB Mouse Bulk kit; Golden Bridge International, Bothell, WA, USA) for 15 min at 37˚C. The sections were then incubated with 3,3-diaminobenzidine (ZSGB-BIO) to visualize the expression of Arg I and iNOS. Positive cells contained brown granules. As a negative control for the LNs from HIV-infected patients, phosphate-buffered saline was used in place of the primary antibody. LN sections from subjects without HIV were treated with primary antibody also served as a control.