Unpolarized Release Enhances Mucosal Responses Figure 1. Immunization schemes. (A) I.n. immunization. (B) Colchicine i.n. immunization for distribution of virus and antigen. (C) Colchicine i.n. immunization for antibody tests. doi:10.1371/journal.pone.0024296.g001 4–10uC by immersing it in a beaker with ice, which was refreshed every 5–10 minutes for 1 hour. Then both the lung and the decalcified nasal turbinate samples underwent serial dehydration and were imbedded in paraffin and cut into slices 2 mm thick. The tissue sections were heated, rehydrated, and subjected to antigen retrieval procedures, including microwaving in 0.01 M citrate buffer (pH 6.0) and incubation in 3% hydrogen peroxide in deionized water. The following experiment was performed using ZSGB-BIO, sp-9001 HistostrainTM-Plus Kits: After tissue sections were blocked for 30 minutes at room temperature, they were incubated with rabbit anti-p24 (1:3000, gift of Dr. Bing Yan, Wuhan Institute of Virology, Chinese Academy of Sciences) for 2 hours at 37uC. Then the tissue sections were incubated with biotin-labeled goat anti-rabbit antibody and strep-HRP separately for 15 minutes at room temperature. They were then visualized using diaminobenzidine (DAB) kit (Maixin-Bio). Before primary antibody incubation, the samples were washed three times with PBS. After primary antibody incubation, they were washed three times with PBS with 0.02% (v/v) Tween 20 and twice with PBS alone. All slides were counterstained with hematoxylin solution. Slides were photographed in an OLYMPUS BX51 microscope equipped with QImaging CCD camera (MiroPublisher 5.0 RTV) and QCapture vision software. Plaque assay The experiment was performed under a biosafety hood using aseptic technique. Vero cells were seeded in 24-well plates and incubated for 24 hours. Serial 10-fold dilutions were inoculated for 2 hours. Inocula were removed and washed with 0.5 ml PBS. Then freshly prepared 1% (m/v) agarose and 26MEM (Gibco, 6% FBS+1% (P+S)) were added. Each plate was incubated for 48 hours, and 0.2 ml of 5 mg/ml crystal violet in 10% formaldehyde was added to fix and stain the cell layers for 2 hours at room temperature. Then the agarose was carefully flushed out with tap water and the plates were air-dried. Plaques were counted. PFU (plaque formation unit) = dilution6P6 (1/V), (P: plaque counts; V: volume of inocula). ELISA (anti-p24 antibody and p24 antigen detection) For anti-p24 antibody detection, 5 mg/ml E. coli recombinant p24 protein was coated in 0.1 M carbonate bicarbonate buffer (pH 9.6) at 37uC for 2.5 hours. Then plates were blocked with 1% BSA in PBS at 4uC overnight. Serial 4-fold dilutions of sample in PBS (0.5% BSA) were added to the plates and incubated for 2 hours at 37uC. Then goat anti-mouse IgG-AP (1:2000, Southern Biotech) or goat anti-mouse IgA-AP (1:2000, Southern Biotech ) in PBS (0.5% BSA) was added and incubated for 1 hour at 37uC. The colorimetric conversion was performed using Sigma FastTM, p-nitrophenylphosphate (PNPP) tablet sets (Sigma, N-2770) for 1 hour at 37uC. After each incubation (but not after blocking), plates were washed with 0.05% Tween 20 in PBS. Plates were evaluated in a microplate spectrophotometer (Multiskan MK3, Thermo LabSystems) at 405 nm and the readouts were calibrated with blanks. Titers of the samples were calculated from endpoint dilutions twice the negative value. For p24 protein detection, the double antibody sandwich method was used. Diluted rabbit anti-p24 poly-antibody (1:500) was coated in 0.1 M carbonate bicarbonate buffer (pH 9.6) at 37uC for 2.5 hours. Then plates were blocked using 10% FBS in PBS at 4uC overnight. Plates containing treated samples were serially 2-fold or 4-fold diluted with PBS containing 10% FBS and 1% (v/v) TritonX-100 and treated for 3 hours at 37uC to release p24 from the HIV-virus-like particle. E. coli recombinant p24 diluted in the same manner was used to create a standard curve to determine the concentration of p24 in samples. Then treated and untreated sample dilutions were placed in wells for PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24296