25 mg/200 μL in PBS and injected i.p. 6 h prior to tissue collection. Sera for ELISA selleck screening library were collected from mice via tail vein bleeds. All experiments were performed according to protocols approved by the UC Davis Animal Use and Care Committee. LN, spleen, and lung tissue cell preparations were generated as previously described 8, 53. Live cells were counted using a hematocytometer and trypan blue exclusion. Cell suspensions were stained as described previously 53 and surface stained

with the following conjugated Ab at previously determined optimal concentrations: CD4/8/F4/80-Pacific Blue (GK1.5/53.6.7/F4/80), CD38-FITC (clone 90), HA-A/PR8-biotin (as described 32), CD1d-Cy5PE (1B1), CD21-Cy55PE (7G6), CD24-Cy55PE (30F.1), and CD23-allophycocyanin (B3.B4) were generated in-house following published protocols (www.drmr.com). C12Id-QDOT605 (23-1 Id 24) was generated using the QDOT Ab conjugation kit (Invitrogen). Commercial reagents used were: CD9-biotin, CD3-Pacific Blue (BD Bioscience), CD40-FITC, CD86-PE, CD44-Cy5PE, SA-Cy7PE (all eBioscience), CD3-allophycocyanin-Alexa750, CD19-Cy5.5allophycocyanin (both Invitrogen), and anti-biotin-PE (Miltenyi Biotec). Live/dead fixable violet staining kit (Invitrogen) was used to discriminate dead cells. For intracytoplasmic C12Id and HA staining cells were fixed for 30 min on

ice using Cytofix/Cytoperm (BD Bioscience), followed by washing and intracytoplasmic staining for 30 min at room temperature in Perm/Wash solution (BD Bioscience). Data acquisition was done using a FACSAria (BD Bioscience) set-up for 13-color analysis 53. Data analysis was conducted

GSK2126458 concentration using FlowJo software (kind gift from Adam Triester, TreeStar). MedLN were fixed in 10% phosphate buffered formaldehyde solution for 24 h and subsequently embedded in paraffin. 4 μm sections were cut using a microtome (Leica). The antigen was retrieved using 10 mM heated citrate buffer (pH 6). Slides were stained overnight at room temperature with biotinylated rat anti-mouse C12 Id and stained for 1 h with biotinylated anti-rat Ab (InnoGenex). For immunohistochemistry staining was revealed stiripentol with ExtrAvidin Phosphatase (Sigma) for 30 min, followed by incubation with NovaRed substrate (Vector). The slide was counterstained with Mayer’s hematoxylin and cover slipped with Permount (Fisher Scientific). Slides for immunofluorescence staining were incubated with the same anti-mouse C12Id Ab for 1 h, then secondary anti-rat Ab (InnoGenex) for 1 h in the dark followed by SA-488 (Invitrogen). After washing, slides were incubated with streptavidin/biotin block (Vector) and the second primary Ab (biotin-conjugated rat anti-mouse CD138 (Syndecan-1), clone: 281-2, BD) was added for 2 h in the dark. After washing, SA-Alexa 568 along with DAPI (both Invitrogen) were added and incubated for 1 h each in the dark. Slides were cover-slipped with an antifade mounting media (ProLong Antifade Kit (P-7481) Invitrogen).