Lsm 5 pascal microscope

For Scanning Electron Microscopy (SEM) images, cathode samples were collected and fixed during 5 hours at room temperature with a 0.1 M phosphate buffer at pH 7.0 containing 2.5% glutaraldehyde. Samples were then washed with the buffer solution without glutaraldehyde before being immerged successively in different concentration of ethanol and acetonitrile as described previously. Nitrogen-dried samples were observed with a Quanta 200 FEG scanning electron microscope (FEI) at an accelerating voltage of 10 kV under high vacuum condition. For Confocal Laser Scanning Microscopy (CLSM) images, RGO paper and carbon paper biocathodes were removed from the MES reactor and were stained with the LIVE/DEAD® BacLight™ Bacterial Viability Kit (ThermoFisher Scientific) as described previously^{43 (link)}. CLSM image were taken with a Zeiss LSM 5 Pascal microscope and analyzed with the ZEN imaging software (Zeiss, Germany).

Sa-β-Gal expression was analyzed as previously described [41 (link)]. Images were acquired using LSM5 Pascal microscope (Carl Zeiss Microscopy).

Cells were fixed with PFA 4% directly on the wells of 12, 48 or 96 well plates for 20 min, washed 3 times with DPBS 1× (ThermoFisher). For the staining, cells were incubated in blocking solution (DPBS 1× with 0.1% Triton X-100 plus 5% Donkey serum) for two hours at room temperature. The corresponding primary antibodies were diluted at suitable concentration in blocking solution, and incubated overnight at 4C. The primary antibodies used are represented in (Additional file 1: Table S1). Cells were washed three times with DPBST (DPBS 1× + 0.1% Triton X-100) and suitable secondary antibody was added in blocking solution for 1 h at room temperature. Then cells were washed three times with DPBST and incubated with DRAQ5 or Hoescht 33342 (1 μg/mL, diluted in DPBS 1×) for 10 min at room temperature for nuclear counterstain. Cells were visualized using an inverted fluorescence microscope (Olympus IX71 microscope) or a confocal microscope (Zeiss LSM5 Pascal microscope) under 10×, 20× or 63× magnification. See Additional file 1: Table S1 for complete details of antibodies used in this study.

Peritoneal macrophages (3 × 10⁴/ well) from C57BL/6 mice were placed in 12-well slides and grown overnight at 37 °C. First, nonadherent cells were removed, and 50 ng/mL mrIL-4 was added. Next, the cells were incubated at 37 °C for 48 h. After fixation with 2% paraformaldehyde–PBS, the cells were washed with PBS three times and blocked with PBS that contained 10% FCS (Invitrogen, Waltham, MA, USA) (FCS-PBS) for 1 h at room temperature. Next, they were incubated with 5 mg of total protein extract from the CD-tryps in FCS-PBS for 1 h at 37 °C, and the cells were then washed in PBS and incubated overnight at 4 °C with IgG anti-MASP49 antibodies (10 µg/mL) and rat anti-mMGL (10 µg/mL) (Santa Cruz, Dallas, TX, USA) diluted in 0.5% BSA-PBS. Finally, Alexa Fluor 594-conjugated donkey antirat or Alexa Fluor 488-conjugated donkey antirabbit (Invitrogen, Waltham, MA, USA), at a 1:250 dilution, was added. The slides were examined under a confocal LSM 5 Pascal microscope (Carl Zeiss, Oberkochen, Germany) [36 (link)].

After incubation with the cells, SPIONs were detected with the help of Perls’ Prussian blue stain, which is a standard indicator for iron ions [43 (link),44 (link)]. The samples were washed from unbound nanostructures using PBS and then fixed according to the standard protocol. Then, the samples were incubated in a solution of 4% HCl/4% potassium hexacyanoferrate (II) (1:1) for 20 min. The samples were washed and stained with Neutral red 0.05% dye for 20 min and, then, washed with PBS. Transmitted light microscopy images of cells with nanoparticles were obtained with a LSM 5 PASCAL microscope (Carl Zeiss, Jena, Germany).