L., Weyand A., Bickley D., Jones D., Whitfield J., Reddy P., Levine J. Histology Tissues of 6H05 interest (liver, lungs, spleen, cervical and mesenteric LNs, thymus, small intestine, Peyer’s patches, skin from your abdomen, back, and paws, and lesions, where relevant) were collected at autopsy. Samples were fixed in formalin (PBS with 4% formaldehyde) overnight, dehydrated, and embedded in paraffin. Tissue sections (4 m) were stained with H&E and safranin and evaluated under light microscopy by an expert pathologist (L.S.). Immunohistochemical stainings were performed on cryo-preserved tissue samples, which were embedded in Tissue-Tek O.C.T. compound (Sakura, Japan; http://www.sakura.com/) and frozen immediately in a bath of liquid 6H05 N2 before storage at ?80C. Tissue sections (4 m) were fixed on glass slides using acetone. Nonspecific binding was blocked using 1.25% BSA (Sigma-Aldrich) before addition of supernatants of OX1, OX8, OX39, 3.2.3, and W3/25 (diluted with an equal volume of BSA solution) for 120 min at ambient temperature. Sections were incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG (Molecular Probes, Invitrogen, Carlsbad, CA, USA) secondary antibody, together with Alexa Fluor 488-conjugated Isolectin B4 (Molecular Probes, Invitrogen) for 60 min at ambient temperature. Between incubations, slides were washed twice in PBS for 5 min, dipped in distilled water and allowed to dry, and finally, mounted in 6H05 polyvinyl alcohol. Images were captured using a Nikon Eclipse E800 fluorescence microscope (Nikon Instruments, Melville, NY, USA; http://www.nikoninstruments.com/) equipped with Nikon Plan-Fluor objective lenses and a F-VIEW digital camera controlled by AnalySIS 3.2 software (Olympus Soft Imaging Solutions, Germany; http://www.soft-imaging.net/). Flow cytometry PB samples of 0.5 mL were collected under analgesia from the lateral tail vein using heparinized Hemato-Clad hematocrit tubes (Drummond Scientific, Broomall, PA, USA; http://www.drummondsci.com/). PBMCs were labeled with combinations of fluorochrome-conjugated anti-rat mAb (summarized in Table 2). Peridinin chlorophyll-conjugated Streptavidin 6H05 (BD PharMingen) was added for secondary staining of biotinylated antibodies. For intracellular staining, buffer solutions from eBioscience 6H05 (San Diego, CA, USA; http://www.ebioscience.com/) were used for fixation and permeabilization of cells, followed by staining with anti-mouse/rat FoxP3 mAb (eBioscience). Immunostained cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences, San Jose, CA, USA) and further analyzed by FlowJo software (Tree Star, Ashland, OR, USA; http://www.treestar.com/). Table 2. Antibodies Used for Flow Cytometric Characterization of T and NK Cell Chimerism test, unpaired, two-tailed), according to Levene’s test of equality of variances. All statistical analyses were performed using PASW Statistics 17.0.2 software (SPSS, Chicago, IL, USA; http://www.spss.com/). RESULTS MHC-mismatched BMT and DLI cause pathological changes typical of aGVHD Clinical BMT across MHC disparities requires extensive T cell depletion of the graft to avoid GVHD and high numbers of hematopoietic cells to overcome the MHC barrier [27]. DLI may be given as therapeutic intervention to improve donor chimerism and to pre-empt or treat neoplastic relapse by way of the graft-versus-tumor effect [28]. With this rat model for alloHCT, we aimed to study the setting of donor-recipient MHC mismatch by using high-intensity conditioning, high doses of T cell-depleted BM, and later treatment with DLI. Lethally irradiated BN rats were transplanted with 30 106 Rabbit Polyclonal to HSF1 T cell-depleted BM cells from PVG.7B donor rats with full MHC mismatch, and were injected with mature T cells from LNs of the same donor strain 14 days after transplantation to invoke aGVHD. We titrated the dose of DLI required to induce aGVHD.