Anti rat af488

To quantify various populations of DENV positive cells in the spleen, spleen tissue was processed to a single cell suspension using collagenase treatment. Surface staining of DCs, macrophages, endothelial cells and reticular fibroblast cells was done using Pacific blue-conjugated anti-cd11c, PE-conjugated anti-cd11b, APC-conjugated anti-cd31, and the primary antibody ERTR7. The secondary antibody anti-rat AF488 (Thermo Scientific) was used to detect ERTR7. Cells were fixed using 4% paraformaldehyde prior to intracellular staining. For intracellular staining of DENV infected cells, cells were permeabilized using 0.1% saponin and primary antibody against DENV non-structural protein 3 (NS3, Genetex) was used. Anti-rabbit AF594 was used as a secondary stain to detect NS3. Flow cytometry was performed using multicolor BD LSRFortessa Analyzer (BD Biosciences) and data was analyzed using FlowJo software.

Kidney tissues were obtained from percutaneous needle biopsy in cold PBS. Tissues were mounted on Surgipath FSC22 Frozen Section Compound (Leica, IL, USA) and stored at − 80 °C until the section of the tissues. As the first step for staining, tissues were fixed with cold acetone. After blocking, primary and secondary antibodies were applied to tissue section and incubated for an hour at room temperature, respectively. Coverslip was mounted on the tissue slides after application of ProLong™ Diamond Antifade Mountant with DAPI (Life Technologies, OR, USA) applied to the tissue slides. The antibodies used for the staining were as follows: anti-c-Kit antibody (host: rabbit, polyclonal) from Biorbyt (Cambridge, UK), anti-CD3 antibody (rat, polyclonal) from Abcam (Cambridge, UK), and anti-rabbit AF594 (donkey, polyclonal) and anti-rat AF488 (donkey, polyclonal) from ThermoFisher Scientific (IL, USA). Slides were imaged by confocal microscopy, FV3000 (Olympus, Tokyo, Japan) and analyzed with FV10-ASW 4.0 Viewer (Olympus, Tokyo, Japan).

Spleens were frozen in OCT compound (Tissue-Tek) prior to sectioning to 10μm thickness using a cryostat (Leica). Sections were acetone fixed at 4°C for 20 min, allowed to dry, then re-hydrated with PBS containing 1% BSA. Sections were stained with primary antibodies, including ER-TR7 (Abcam) for FRCs and with an antibody against DENV NS3 (GeneTex). After washing with PBS, secondary antibodies including anti-rabbit AF546 and anti-rat AF488 (Thermo Scientific), were incubated for 2h at room temperature prior to washing. Slides were mounted with Prolong gold antifade reagent (Thermo Fisher Scientific). Images were acquired using a Carl Zeiss LSM710 confocal microscope. ImageJ software was used to generate the colocalization images.