Fitc conjugated anti mouse secondary antibody

BG2-c2 cells were transfected with pAc-Myc-tkv-Flag or pAc-Myc-wit-Flag in the presence or absence of gef26 dsRNA. At 72 h post-transfection, live cells were incubated with an anti-Myc antibody (1:200, Cell Signaling) at 4 °C for 1 h to label Myc-Tkv-Flag or Myc-Wit-Flag proteins expressed on the cell surface, followed by incubation at 25 °C for 10 min to allow internalization of the labeled receptors. Cells were subsequently washed in an ice-cold acidic buffer (0.5 M NaCl, 0.2 M acetic acid, pH 4.0) for 15 min to remove any remaining bound anti-Myc antibody and fixed in PBS containing 4% formaldehyde for 10 min. Fixed cells were permeabilized in PBT-0.2 (PBS, 0.2% Triton X-100) for 10 min, blocked with PBS containing 1% BSA for 1 h, and sequentially incubated with a mouse anti-Flag primary antibody (1: 500, Sigma-Aldrich) and a FITC-conjugated anti-mouse secondary antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) in PBS containing 1% BSA. Stained cells were mounted with SlowFade antifade medium (Invitrogen) and imaged with a LSM 800 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) using a Plan Apo 63 × 1.4 NA oil objective. The number of intracellular Myc-positive puncta was measured in cells with similar fluorescence intensities of Flag staining.

The effects of triptolide on the expression of adhesion molecules, including ICAM-1 and VCAM-1, were determined using cell-based ELISA assay as previously described [20 (link)]. In brief, HUVECs in 96-well plates were fixed with 4% paraformaldehyde for 5 min and rinsed 3 times with PBS following treatment. The fixed cells were permeabilized with pre-chilled MeOH for 10 min at -4 °C, followed by blocking with PBS containing 1% BSA and 0.2% triton X-100 for 1 h. Subsequently, the cells were incubated with mouse monoclonal antibody against ICAM-1 or mouse monoclonal antibody against VCAM-1 (dilution, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 12 h. Following washing, the cells were incubated with FITC-conjugated anti-mouse secondary antibody (dilution, 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. Incubation with PBS, instead of the primary antibodies, was used as a negative control. The optical density of each well was determined by the use of a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 485/520 nm. The optical density of the negative control was subtracted as background from that of each well.

Paraffin sections of right lung tissues were washed three times with PBS. After fixation with 4% paraformaldehyde (PFA) and 0.3% Triton X-100 for 20 min, the tissue sections were incubated with an anti-HO-1 (1 : 100) (Abcam, USA) primary antibody at 4°C overnight. The next day, the slides were incubated with a FITC-conjugated anti-mouse secondary antibody (1 : 100) (Jackson ImmunoResearch, USA) for 1 h at 25°C. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were observed by using a fluorescence microscope (#BX53, OLYMPUS, Japan).

Cells were seeded on 12-well culture plates that contained chamber slides and treated with IR or left untreated. Briefly, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After fixation, cells were lysed in 0.2% Triton X-100 for 10 min and incubated with an anti-RIP1 antibody (MBL, Nagoya, Japan) overnight, followed by a FITC-conjugated anti-mouse secondary antibody (Jackson Immuno Research, West Grove, PA, USA) for 2 h at room temperature. Nuclei were stained with mounting solution and images of stained cells were acquired under a LSM880 confocal microscope (Carl Zeiss, Germany).