Rat igg2b alexa fluor 488

Biopsies were embedded in OCT Tissue TEK and stored in liquid nitrogen. Frozen sections were cut (5 μm) using a cryostat, after which sections were stored at −80 °C until further use. For staining, sections were thawed and air-dried at room temperature and subsequently fixed with acetone [34 (link)]. Sections were washed and stained with monoclonal primary antibodies or isotype controls: rat anti-human CXCR5-Alexa Fluor 488 IgG2b (BD Biosciences), rabbit anti-human Bcl-6 IgG (Bio SB, Santa Barbara, CA), mouse anti-human ICOS IgG1 (Novo Biologicals, Littleton, CO, USA), rat IgG2b Alexa Fluor 488 (isotype control, BD Biosciences), mouse IgG1 (isotype control, Dako Cytomation, Heverlee, Belgium), or rabbit IgG (isotype control, Dako Cytomation) diluted in PBS/1% BSA/10% normal human serum (NHS; Lonza, Basel, Switzerland) overnight at 4 °C. After washing with PBS, (directly labeled) secondary antibodies (goat anti-rabbit IgG Alexa 546 and goat anti-mouse IgG1 Alexa 633, both from Life Technologies, Bleiswijk, The Netherlands) were incubated for 30 min in PBS/1%BSA/10% NHS. After washing with PBS, slides were covered with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed on a confocal imaging microscope (Leica Microsystems, Wetzlar, Germany).

PB was collected in heparin-coated tubes (Venosafe plastic tubes, Terumo Europe N.V., Leuven, Belgium) and PB mononuclear cells (PBMC) were isolated using high density centrifugation (Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×10⁶ cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively (BD Biosciences, Erembodegem, Belgium). B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 (all from BD Biosciences, Erembodegem, Belgium). Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 (all from BD Biosciences, Erembodegem, Belgium), CD25 PerCP-Cy5.5 and CD127 PE (eBioscience, San Diego, USA). Following isotype controls were used: mouse IgG1 PErCP-Cy5.5, IgG1 PE, IgG1 PE-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 PE-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 (all from BD Biosciences, Erembodegem, Belgium). All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).