Ncl l cd3 565

Tissue sections (skin and fat) were stained with markers for macrophages (CD68, DAKO/Agilent M0814, CA, USA), mast cells (CD117, BioCare CM5296C, CA, USA), T lymphocytes (CD3, Leica NCL-L-CD3-565, IL, USA), endothelial cells (CD31, DAKO/Agilent M0823, CA, USA), blood vessels (SMA, Thermo MS-113-P, MA, USA), and lymphatic vessels (D2-40, Biolegend SIG-3730, CA, USA), counterstained with hematoxylin; positive and negative controls were included (San Diego Pathologists Medical Group, CA, USA). Angiogenesis was assessed by Masson's trichrome stain (Sigma kit HT102A-1KT, MO, USA). All sections were coded and scored in a blinded manner by two independent investigators. The quantification of CD3, CD68, and CD117 positive cells was expressed as the average of total number of cells counted in 10 fields at 20x magnification (five fields in skin and five in fat).

For immunodetection of CD20, CD3, and p24, formalin-fixed and paraffin-embedded (FFPE) sections from human lymph nodes of HIV-infected patients were dewaxed and placed in decreasing ethanol concentrations. Heat-induced epitope retrieval was performed by autoclaving at 120 °C for 15 min in a citrate buffer pH 6 (Abcam). Then, the slides were permeabilized in 1X Tris-buffered saline (TBS) (Fisher scientific) with 0.1% Triton X-100 (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) for 10 min. Subsequently, blocking was added for 2 h with 1X TBS supplemented with 10% normal donkey serum (Jackson Immunoresearch) and 1% BSA. The slides were incubated with primary antibodies overnight at 4 °C: anti-CD20 (goat-polyclonal anti-CD20 antibody, 1/100, Abcam ab194970), anti-CD3 (mouse-monoclonal anti-CD3 antibody, 1/100, Leica Biosystems NCL-L-CD3-565), or anti-p24 (mouse-monoclonal anti-p24 antibody, 1/10, Dako-Agilent M0857), diluted in TBS 1 × −1% BSA. Next, samples were washed and incubated for 1 h with the appropriate secondary antibody: Alexa Fluor 546 donkey anti-goat (Invitrogen, 712-586-150) and Alexa Fluor 647 donkey anti-mouse (Invitrogen, A-31571), counterstained with DAPI (4′,6-diamidino-2-phenylindole-dilactate, ThermoFisher), and mounted with Fluoromount G (eBioscience).