Fitc conjugated secondary rabbit anti rat igg

P. falciparum IEs were DNA-labeled with ethidium bromide and surface-labeled with rat anti-PfEMP1 antibody (anti-HB3VAR21-DBLβ_D4 or anti-HB3VAR34-DBLβ_D4 antibodies; 1:20) and FITC-conjugated secondary rabbit anti-rat IgG (1:150; Vector Labs) as described previously (Joergensen et al., 2010 (link)). Fluorescence data from ethidium bromide–positive cells were collected on an FC500 MPL flow cytometer (Beckman Coulter) and analyzed using WinList, version 9.0 (Verity Software House). hCMEC/D3 and human brain microvascular pericytes were surface-labeled for CD31 and NG2, respectively. Human astrocytes were permeabilized with 0.1% Triton X-100 before staining with glial fibrillary acidic protein. Fluorescence data were collected on a CytoFlex S flow cytometer (Beckman Coulter) and analyzed using FlowJo (Becton Dickinson).

P. falciparum IEs were DNA labeled with ethidium bromide and surface labeled with rat anti-HB3VAR21-DBLβ_D4 (1:20) and fluorescein isothiocyanate (FITC)-conjugated secondary rabbit anti-rat IgG (1:150; Vector Labs) as described previously (50 (link)). Fluorescence data from ethidium bromide-positive cells were collected on an FC500 MPL flow cytometer (Beckman Coulter) and analyzed using WinList, version 9.0 (Verity Software House).

P. falciparum IEs were DNA-labelled with ethidium bromide and surface-labelled with rat antibodies (1:40) specific for 3D7 PFD1235w, IT4VAR13, and HB3VAR03 as described (26), followed by FITC-conjugated secondary rabbit anti-rat IgG (1:150, Vector labs). Fluorescence FITC data from ethidium bromide-positive cells were collected on a Cytometic FC500 MPL flow cytometer (Beckman Coulter), and analysed using Winlist software (Verity House).
Resting or TNF-α-activated (10 ng/mL overnight) BOECs between passage 3–6 were detached with trypsin-EDTA, washed with DPBS, and suspended in FACS buffer (DPBS plus 3% BSA). The cells were incubated (45 min; 4˚C) with primary antibody, washed with assay PBS plus 3% BSA, incubated (30 min; 4˚C) with FITC-conjugated secondary antibody, and analysed by flow cytometry as above. Mouse monoclonal antibodies against human MUC18 (CD146; clone P1H12, Biolegend), PECAM-1 (CD31; clone 9G11, R&D systems), ICAM-1 (CD54; clone BBIG-11, R&D systems), thrombomodulin (CD141; clone 501733, R&D systems), platelet glycoprotein 4 (CD36; clone FA6.152, Beckman Coulter), and goat polyclonal antibody against human EPCR (CD201; polyclonal R&D systems) were used as primary antibodies. FITC-conjugated anti-mouse (Vector Laboratories) and anti-goat (Vector Laboratories) antibodies were used as secondary reagents. In addition, FITC-conjugated mouse anti-human CD133 was used.