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The samples were fixed with 2% osmium tetroxide in 0.1?M phosphate buffer at 4��C for 2?hr and were then dehydrated through a series of graded ethanol wash steps for scanning electron microscopy. The samples were transferred into tert-butyl alcohol three times for 30?min each and frozen at 4��C. After drying, the samples were coated with a thin layer (35?nm) of osmium with an osmium plasma coater (NL-OPC80NS, Nippon Laser & Electronics Laboratory). The samples were observed with a scanning Crenolanib in vitro electron microscope (S-800, Hitachi) at an acceleration voltage of 10 kV, and images were captured with a digital camera. Consecutive cryostat colon sections were used for immunohistochemistry with purified rat anti-CD4 (RM4-5; BD PharMingen), rat anti-CD11b (M1/70; Abcam) and rabbit anti-GFP (green fluorescent protein; MBL) mAb. In brief, tissue samples were fixed in PBS containing 4% paraformaldehyde for 2?hr and were then placed in PBS containing 15% sucrose overnight, replaced in PBS containing 30% sucrose overnight, mounted in optimal cutting temperature (OCT) embedding compound, and frozen at ?80��C. OCT-embedded tissue samples were cut into 10-��m-thick RecBCD serial sections, placed on coated slides, and fixed with 2% paraformaldehyde phosphate buffer solution for 5?min. Slides were then incubated with the primary antibody at room temperature for 2?hr and stained with Alexa Fluor 568 goat anti-rat?IgG (Invitrogen) for CD4 and CD11b detection and with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) for GFP detection at room temperature for 2?hr. All slides were counterstained with Hoechst (Invitrogen) and observed by confocal microscopy. For surface and intracellular buy Antidiabetic Compound Library staining, we used the same protocol as previously described (Atarashi et?al., 2011) (Supplemental Experimental Procedures). FITC-, PE-, PerCP-Cy5.5-, APC-, PE-Cy7-, APC-Cy7- or Alexa Fluor 647-conjugated mAbs against CD11c (HL3), CD3 (145-2C11), and CD4 (RM4-5) were from BD Biosciences, CD11b (M1/70), F4/80 (BM8), Foxp3 (FJK-16S), and IL-10 (JES5-16E3) were from eBioscience, and CD25 (PC61) was from BioLegend (Supplemental Experimental Procedures). A mouse inflammatory cytometric bead array (CBA) kit (BD Biosciences) was used for cytokine measurements, according to the manufacturer��s instructions. Samples were analyzed with a FACSCanto II flow cytometer (BD Biosciences). Bacterial genomic DNA was extracted from fecal pellets with phenol-chloroform extraction as previously reported (Matsuki et?al., 2004). qPCR analysis was performed with a Thermal Cycler Dice Real Time System TP800 (Takara). The primer sets used in this study are described in the Supplemental Experimental Procedures. The results are expressed as mean?�� SEM. Groups of data were compared with the Mann-Whitney U test, Tukey-Kramer test, and Student��s t test. Differences were considered to be statistically significant when p?