Gfp fitc

Splenocytes were suspended in staining media (SM: 3% FCS, 5 mM EDTA, 0.05% sodium azide, PBS) with the FcR blocking antibody, 2.4G2 and ethidium monoazide (EMA) for live/dead discrimination. Reagents used for staining were either prepared in house: 4-44-biotin, PDCA1 (927)-Alexa 647, CD62L (MEL-14)-Alexa 647; or purchased from Biolegend: CD44 (1M7)-APC/Cy7, CD11c (N418)-PE/Cy7, CD4 (GK1.5)-PE/Cy7, Gr1 (RB6.8C5)-biotin; from BD: CD22.2 (Cy34.1)-PE, TCRβ (H57-597)-PE, I-A/I-E (M5/114.15.2)-PE; from eBioscience: CD11b (MAC-1)-PE, streptavidin-PE/Cy7; or from Invitrogen: streptavidin-PacBlue. Surface stained cells were fixed with 1% PFA, washed and resuspended in SM. For intracellular staining, cells were fixed in 4% PFA in BD Perm/Wash Buffer, washed, blocked (with 5% rat serum), and stained with 4-44-Alexa 647 or GFP-FITC (Rockland) in the BD Perm/Wash Buffer, with a final wash in SM. Cytometry was performed on a LSRII (BD) and data analyzed with FlowJo software.

Spleens were snap frozen in OCT tissue-freezing solution and stored at −80°C. Tissues were cut into 6um sections and processed as described previously (22 (link)). Sections were stained with GFP FITC (Rockland Immunochemicals), CD4 (clone RM4-5) FITC, IgD (clone 11-26) Alexa-647 (both from eBiosciences), PNA biotin (Vector Laboratories), and rabbit IgG anti-FITC 488 and Alexa-555 (both from Invitrogen). Images were obtained from a laser-scanning confocal microscope (model 510 META; Carl Zeiss, Inc.) at 25x magnification. ImageJ software from the National Institutes of Health was used for the measurement of GC and B cell follicle size as well as for T cell counting.

Antibodies with the following specificities were used for flow cytometry and histology: GFP-FITC (goat, Rockland), CD38-PE/Cy7 and -Pacific Blue (90, BioLegend), CD45R-PE, -BV421, -PE/Cy7, and -APC/Cy7 (RA2–6B2, BioLegend), Bcl6-AL647 (K112–91, BD Pharmingen), Bcl6-AL647 (7D1, Santa Cruz), Activated Caspase-3-PE (C92–605, BD Pharmingen), BrdU-bi and –AL647 (3D4, Phoenix), CD4-BV421 (GK1.5, BioLegend), ICOS-APC (C398.4A, BioLegend), CD62L-APC/Cy7 (Mel-14, BioLegend), CCR7-PE (4B12, BioLegend), CD86-BV605 (GL1, BD Bioscience), CD40-bi (HM40–3, eBioscience), CXCR4-PE (L276F12, BioLegend), CD83-bi (Michel-19, BioLegend), Mouse IgD-V450 (11–26c.2a, BD Bioscience), and CXCR5-PE (2G8, BD Pharmingen). The following antibodies were purified and conjugated in-house: CD4-Pacific Blue and –AL680 (GK1.5), CD45R-AL647 (RA2–6B2), IgD-bi (11–26c), CD35-bi (8C12), and CD23-AL680 (B3B4). Anti-FITC-AL488 (goat polyclonal, Invitrogen), streptavidin-BV421, and –BV605 (BioLegend), and streptavidin-AL555, and –PECy7 (Invitrogen) were used as secondary reagents.