Horseradish peroxidase hrp labeled goat anti rabbit igg

FNC with a purity of 98.5% and 3TC with a purity of 99.6%, were synthesized by Dr. Jun-biao Chang, Zhengzhou University, China. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), N, N-dimethylformamide (DMF), phytohemagglutinin (PHA-p), interleukin-2 (IL-2), Fc-specific anti-mouse IgG, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG and zidovudine (AZT) were purchased from Sigma-Aldrich company. Raltegravir (RAL) was purchased from Selleck Chemicals. Nevirapine (NVP) was purchased from US Pharmacopeia. Enfuvirtide (T-20) was purchased from Roche Inc. RPMI-1640 media and fetal bovine serum (FBS) were purchased from Invitrogen, USA. FBS was heated at 56°C, 30 min to inactivate complements. Indinavir (IDV) was kindly gifted by Dr. Yu-Ye Li. Ficoll-Hypaque was purchased from Haoyang Biotechnonlogy Inc. Rabbit anti-p24 polyclonal antibody and mouse anti-p24 monoclonal antibody were prepared in our laboratory. Custom primers and fluorophore-labeled probes were synthesized by Invitrogen.

Vero cells cultured in 24-well plates at 100% confluence were infected with 10-fold serial dilutions of the viruses for 1 h, and the supernatant was replaced with fresh DMEM containing 2% FBS and 1% (w/v) Avicel (Millipore). The overlay was removed 5 days post-infection and fixed with 4% formaldehyde. The fixed cells were permeabilized with 0.2% Triton X-100 and probed with an anti-NP antibody and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Sigma, Tokyo, Japan). Cells were stained using a hydrogen peroxide (DAB) kit (Servicebio, Wuhan, China). Wells containing 10–100 immunofocus units (plaques) were selected for imaging and counting.