Rat anti mouse f4 80 antibody

Immunofluorescence staining was performed as described previously (10 (link)). Briefly, freshly frozen renal tissues were fixed with 4% paraformaldehyde, permeabilized in 1% Triton X-100 and then incubated with a blocking buffer. Subsequently, the samples were incubated with rat anti-mouse F4/80 antibody (eBioscience, Inc., San Diego, CA, USA). The slides were exposed to Cy3-labeled secondary antibody (Chemicon, Temecula, CA, USA). The nuclear staining was performed using DAPI. The digital images were captured with a Zeiss Z1 microscope (Carl Zeiss AG, Göttingen, Germany) in 10 randomly chosen, non-overlapping fields at a magnification of ×400.

BAL fluids from FITC-SiO₂ NPs treated mice (5 mg/kg) were recovered 5 h post NP instillation and cells were centrifuged at 4°C (2000 rpm, 10 min). Cells were washed in 1% BSA-PBS buffer and incubated with monoclonal anti-mouse CD16/32 antibody (BD Biosciences) for 15 min on ice to block Fc receptors. Cells were then stained with a rat anti-mouse F4/80 antibody coupled to eFluor450 (clone: BM8, eBioscience) and a hamster anti-mouse CD11c antibody coupled to APC (clone: HL3, BD Biosciences) (30 min, at 4°C in 1% BSA-PBS). After washing, fluorescence associated with cells was acquired in a CyAn ADP cytometer (Beckman Coulter). Analysis of data was performed using FlowJo program. FITC positive cells were only found in the macrophage gate (named R1 in FSC/SSC dot plot). To detect FITC positive cells from negative cells, BAL fluids from non-treated mice were obtained and treated in parallel to determine the auto-fluorescence signal (characteristic of AM) from R1 population.

Fresh mouse dorsal skin tissues were embedded in optimal cutting temperature compound and 6- to 8-μm frozen sections were cut. Then, the frozen tissues or HaCaT cells were fixed in 4% PFA for 15 min. Afterwards, the samples were permeabilized and blocked using blocking buffer (0.2% Triton-X/5% donkey serum) for 1 h. Next, the skin sections were incubated with different antibodies: rat anti-mouse CD4 antibody (1:100), rat anti-mouse CD31 antibody (1:100), and rat anti-mouse F4/80 antibody (1:100), and these antibodies were purchased from eBioscience. HaCaT cells were incubated with rabbit anti-phospho-NF-kB p65 antibody (1:200, Cell Signaling, USA) at 4°C overnight. Anti-rat Alexa 488 (1:200; Invitrogen, USA) and anti-rabbit Alexa 594 (1:200; Invitrogen, USA) were used as the secondary antibody for staining samples 1 h at room temperature. Finally, all samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min to visualize nuclei. Fluorescence images were acquired under a fluorescence microscope (Zeiss, Germany).