Bovine serum albumin (bsa)

Immunofluorescence analysis of porcine MoDCs was conducted as described in our previous reports [15 (link)]. Mounted MoDCs were incubated for 20 min with 1 % bovine serum albumin and 2 % normal goat serum (Vector Laboratories, Burlingame, CA) in PBS at room temperature to block nonspecific binding sites. After removal of the blocking solution, samples were incubated for 16 h at 4 °C in a humidified chamber with one of the following primary antibodies: anti-porcine CD172a-fluorescein isothiocyanate (FITC) SWC3 IgG1 (Southern Biotech), anti-porcine CD11R1-unlabeled mouse IgG1 (AbD Serotec, Kidlington, Oxford, United Kingdom), anti-porcine major histocompatibility complex class II (MHC-II)-unlabeled mouse IgG2a, anti-porcine TLR2-unlabeled rabbit IgG (Santa Cruz); or anti-porcine TLR4-unlabeled rabbit IgG (Santa Cruz). Samples were washed in PBS and then treated with one of the following secondary antibodies: anti-mouse IgG2a-FITC (AbD Serotec), anti-mouse IgG-FITC (AbD Serotec), or anti-rabbit IgG-FITC (Santa Cruz), when necessary.

Retinal cells in 24 well plates (BD Biosciences) were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min, washed for 3×10 min of PBS, blocked in blocking solution (0.5% bovine serum albumin (g/ml), 0.3% Tween-20, 15% normal goat serum (Vector Laboratories, Peterborough, UK) in PBS) for 20 min and incubated with primary antibody (anti-rat βIII-tubulin, raised in mouse, #T8660 diluted at 1∶500 in antibody diluting buffer (ADB; 0.5% bovine serum albumin, 0.3% Tween-20 in PBS) for 1 h at room temperature. Cells were then washed for 3×10 min in PBS, incubated with the secondary antibody (anti-mouse IgG Fluor 488, raised in goat, 1∶400, #A-11001; Life Technologies, Molecular Probes, Paisley, UK) diluted in ADB for 1 h at room temperature, washed for 3×10 min in PBS, mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories) and stored at 4°C.

The identity of isolated and purified SSCs was
verified by tracking the PLZF protein (17 (link)) in the
obtained colonies from the cell suspension. PLZF
protein, as a marker of stem cells, was detected in the
SSC-derived colonies through immunocytochemistry
on day 7 of culturing. In brief, the cells grown
on glass slides were fixed for 20 minutes in 4%
paraformaldehyde at room temperature and then
rinsed with PBS. The cells were permeabilized with
0.2% Triton X-100 (MP Biomedicals, USA) for 1
hour to facilitate antibody penetration, and the slides
were washed with PBS supplemented with 0.2%
bovine serum albumin (Vector Laboratories, USA).
Nonspecific antigens were blocked with 10% normal
goat serum (Vector Laboratories, USA), and the slides
were then incubated overnight at 37°C with a mouse
monoclonal anti-human PLZF antibody (diluted 1:100,
Santa Cruz Biotechnology, USA). The slides were
washed with PBS and secondary antibody (goat Texas
red-conjugated anti-mouse IgM, diluted 1:100, Sigma,
USA) was applied for 2 hours at room temperature in
the dark.

Boiled cell lysates (10 μl) were loaded onto a NuPAGE 4–12% BisTris SDS-polyacrylamide gel (Invitrogen) and then transferred to polyvinylidene fluoride membranes (VWR) for 1 h at 100 mV. Blots were blocked using 5% bovine serum albumin (US Biologicals) in 50 mm Tris, pH 8.0, containing 150 mm NaCl and 0.5% Tween buffer (TBST) for 2 h at 4 °C. Biotinylated anti-maltose-binding protein antibody (Vector Laboratories) was diluted 1:1500 in 1% bovine serum albumin in TBST and incubated overnight at 4 °C. Blots were washed with TBST for 1 h with three buffer exchanges at room temperature before incubation with secondary antibody in 1% bovine serum albumin in TBST at 1:1500 dilution, peroxidase-conjugated streptavidin (Jackson ImmunoResearch) for 2 h at room temperature. Blots were again washed for 1 h using three exchanges of TBST. A 1:1 mixture of buffers from the Pierce ECL Western blotting substrate kit was added to the blot, and chemiluminescence was measured using an ImageQuant LAS 4000 (GE Healthcare).

Trachea specimens from necropsy were embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) and immunostained as previously described [30] (link) with the following modifications: sections were fixed in acetone, blocked in bovine serum albumin (Vector Laboratories, Burlingame, CA) and stained with a FITC-conjugated mouse anti-human IL-6 (clone MQ2-13A5; eBiosciences, San Diego, CA). A FITC-conjugated mouse IgG1 was used as an isotype control (eBiosciences). Both control and IL-6 antibodies were used at a concentration of 10 µg/ml with an overnight incubation.

Zebrafish expressing DsRed under the control of the cmlc2 promotor^{62 (link)} were treated with atorvastatin or DMSO from tailbud stage on and fixed at 48 hpf with 1% formaldehyde for 50 min at room temperature (RT). After washing with PBS, embryos were incubated for 1 h in blocking solution containing 10% normal goat serum (Vectorlabs, UK), 2 mg/ml bovine serum albumin (BSA) and 0.2% saponin in phosphate-buffered saline (PBS). Primary antibodies diluted in PBS with 0.2% saponin were added to the embryos and incubated overnight at 4 °C. An antibody against DsRed was used to enhance the fluorescence of the DsRed. Atrial myosin was stained with the S46 antibody to distinguish atrial from ventricular cardiomyocytes. Next day, samples were washed several times for 15 min with PBS supplemented with 0.2% saponin and subsequently incubated in secondary antibodies (diluted in blocking solution) for 3 h at RT, followed by several washing steps with PBS. After mounting the embryos with Vectastain containing 4′,6-diamidino-2-phenylindole (DAPI; Vectorlabs) between coverslips, samples were stored at 4 °C until imaging via confocal microscopy.

Paraffin-embedded samples of 5-µm thickness were prepared as for H-E staining. Antigen retrieval was carried out using HistoVT One (Nacalai Tesque, Kyoto, Japan) at pH 7.0, 70°C for 20 min; blocking was carried out using 1% bovine serum albumin (Vector Laboratories, Inc., CA) at room temperature for 60 min. The samples were subsequently reacted with the primary antibody overnight at 4°C. They were then reacted with fluorescence-labeled secondary antibody in the dark at room temperature for 60 min, Proliferating cell nuclear antigen (PCNA) as proliferation maker, TdT-mediated dUTP nick end labeling (TUNEL) as apotosis maker and 8-hydroxy-2'-deoxyduanosine (8-OHdG) as oxidative maker, nuclear stained (ProLong^® Gold antifade reagent with DAPI, Molecular Probes^TM, OR,), and mounted. Fluorescent immunohistochemical observation was carried out using a fluorescence microscope (IX71, Olympus, Tokyo, Japan).