Ten animals were infected with 5 × 106 red blood cells parasitized by P. berghei-NK65 (PbNK-65) or only injected with saline (negative control group). After 14 days of infection, control and infected mice were
anaesthetized, killed and their thymi were collected and used in the experiments described below. Thymi were minced, washed PI3K inhibitor and resuspended in phosphate-buffered saline (PBS) containing 5% fetal calf serum for subsequent cellularity evaluation, which was followed by triple immunofluorescence staining. Appropriate dilutions of the following fluorochrome-labelled monoclonal antibodies were used: fluorescein isothiocyanate (FITC)/anti-CD4 (clone GK1.5), Alexa Fluor 647/anti-CD8 (clone 53-6.7), PeCy-7/anti-CD3 (clone 145-2C11), phycoerythrin (PE)/anti-CD49d (clone 9C10), PE/anti-CD49e (clone 5H10-27), PE/anti-CD49f (clone GOH3), PE/anti-CXCR4 (clone B11/CXCR4) and PE/anti-CCR9 (clone 242503). Selleck FDA-approved Drug Library These reagents were purchased from Pharmingen/Becton-Dickinson (South San Francisco, CA) and R&D Systems (Minneapolis, MN). Fluorochrome-labelled isotype-matched negative controls for the specific monoclonal antibodies were obtained from Pharmingen. Cells were stained for 20 min and then washed with PBS, fixed and analysed by flow cytometry in a FACsCANTO® device (Becton-Dickinson) equipped with
Diva software. Analyses were performed after recording 10 000 events for each sample using FCS Express V3 software (BD Biosciences, San Jose, CA). Splenic cells from infected and control animals were also processed and analysed very by flow cytometry. In this case, CD4+ and CD8+ cell populations were analysed by gating on CD3+ cells. Thymi were embedded in Tissue-Tek (LEICA Instruments,
Nussloch, Germany) and subsequently frozen at −70°. Five-micrometre thick cryostat sections were settled on silanized glass slides, acetone-fixed and blocked with PBS/1% bovine serum albumin (BSA). Samples were submitted to anti-fibronectin or anti-laminin primary antibody incubation (Sigma-Aldrich, St Louis, MO) for 1 hr at room temperature, washed three times with PBS and labelled with FITC-coupled secondary antibody incubation (Santa Cruz Biotechnology, Santa Cruz, CA) for an additional 30 min. Samples were analysed by fluorescence microscopy (Olympus) and the images obtained were subsequently quantified for the presence of ECM proteins using the Image J software.19 The expression of chemokine genes was evaluated by real-time quantitative transcription polymerase chain reaction (RT-qPCR). Thymus RNA was extracted from tissues using the Illustra RNAspin Mini (GE Healthcare, Amersham, UK). After RNA quantification and analysis of RNA integrity on a 1·5% agarose gel, reverse transcription was performed with approximately 2 μg of RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions.