Experiments were performed with 6- to 8-wk-old BALB/c, C.B-17 SCID 41, LTα−/−28, and CXCR5−/− mice 27. C57BL/6 mice were used as controls. BALB/c mice were immunized
i.p. with 100 μg of alum-precipitated 2-phenyl-5-oxazolone coupled to the carrier protein chicken serum albumin and spleens were analyzed 7 (early) and 15 (late GC) days later 42. Animal experiments were approved by the institutional animal care and use committee. For FACS analysis PE-anti-CD23 (B3B4) (BD Pharmingen), biotinylated PNA (Vector), Cy5- and Alexa 488-conjugated anti-CD21 (7G6), biotinylated anti-B220 (RA3-6B2) and anti-CD4 (GK1.4) Ab (provided by the DRFZ) were used. The FDC network was stained with the biotinylated Ab M2 (Immunokontact). Biotinylated Ab were visualized with
Alexa 488 or Cy5 coupled to streptavidin (Molecular Probes and BD Biosciences), unlabelled Ab by secondary anti-rat-Alexa buy Buparlisib 647 or anti-rabbit-Rhodamine-X Ab (Molecular Probes). Biglycan (LF-159) specific Ab were a generous gift from L. W. Fisher (Bethesda, MD, USA) 43 and BP3 specific Ab from M. Cooper (Emory University, USA) 19. Spleens were dissected, embedded in OCT compound (TissueTek), snap frozen and stored at −70°C. For immunohistology, tissue sections (8 μm) were air dried, fixed in acetone and stored at −20°C; for LCM sections were stored without fixation at −70°C. For immunostainings, Src inhibitor sections were thawed, blocked with 3% BSA in PBS and incubated with specific Ab. From each of the BALB/c mice analyzed, one half of the spleen was used for dissection of FDC and the other half for preparation of B cells. Splenic single cell suspensions were labeled with B220 specific Ab and B cells enriched by MACS. Follicular B220+CD21intCD23+ and GC (B220+ PNAhi) B cells were sorted to
high purity (>99%) using FACS Aria (BD Biosciences). Before staining, sections were rapidly thawed, fixed for 30 s in 75% ethanol and washed twice for 5 s in RNase-free buffer (HistoGene Arcturus). To visualize the network of FDC, sections were incubated for 90 s at 4°C with anti-CD21-Alexa 488 (20 μg/mL). To distinguish between primary and secondary follicles, consecutive sections were stained with PNA coupled to RhodamineX (Vector). Splenic tissue sections from SCID mice were stained with BP3-specific Ab labeled with Alexa 488 (20 μg/mL). Sections were dehydrated in graded ethanol and cleared in xylene. Both, FDC networks Metalloexopeptidase and BP3hi stromal cells were dissected (approximately 1 mm2) using LCM (Veritas, Arcturus). From each cell preparation (approximately 20 000 cells each) RNA was extracted (PicoPure RNA isolation Kit, Arcturus), mRNA amplified and labeled in two cycles (RiboAmp OA RNA Amplification Kit, Arcturus, GeneChip 3′-amplification for IVT labeling Kit, Affymetrix). For each cell population and each time point, two independent RNA preparations were analyzed. The integrity of isolated total RNA and amplified cRNA was assessed using the Agilent 2100 Bioanalyzer.