Alexa fluor 555 conjugated anti rabbit igg secondary antibody

HeLa cells grown in 8-well LabTek dishes were transfected with GalT-mMaple3 or GalT-moxMaple3 using Lipofectamine2000 as per manufacturer’s recommendations. 16 h post-transfection, cells were fixed in 4% paraformaldehyde in 1x PBS for 20 min. Cells were then permeabilized with 0.1% triton X-100 in 1x PBS. Cells were blocked in RPMI1640 containing 10% FBS for 30 min. Cells were incubated with anti-GM130 (anti-GM130, BD Transduction Laboratories catalogue number 610823, 1:500 BD Biosciences, San Jose, CA) followed by Alexa Fluor 555 conjugated anti-rabbit IgG secondary antibody (catalogue number A21428 used at 1:500, Life Technologies) in the RPMI block solution. Cells were imaged in 1x PBS on a ZEISS LSM880 AxioObserver with a EC Plan-Neofluar 40x oil immersion objective (NA:1.3) using 488 nm and 561 nm laser excitation through a MBS 488/561 nm dichroic and a BP495–550 + LP570 nm emission filter. z-stacks contained 34 slices (1136 × 1136 px) in 0.2 µm intervals and were subject to SR-Airyscan processing (ZEISS ZEN Black) and maximum intensity projection. Linear contrast enhancement was performed in Fiji software (http://fiji.sc/Fiji).

Cells were seeded on glass coverslips (22 × 22 mm, Matsunami, Tokyo, Japan) placed in 35-mm dishes. After 24 h incubation, cetuximab (10 µg/ml) treatment was performed for 10 days. All samples were fixed in 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed extensively with PBS. The cells were incubated at 37 ºC for 2 h with the following primary antibodies: rabbit anti-p27^Kip1 (ab32034, Abcam PLC) to evaluate accumulation of the p27 protein. In addition, DAPI (1 µg/ml, Sigma-Aldrich, St. Louis, MO, USA) was used for nuclear staining. Slides were then incubated for 1 h with an Alexa Fluor 555-conjugated anti-rabbit IgG secondary antibody (A32732, Life Technologies, CA, USA). The slides were mounted using PBS containing 10% glycerol and sealed with glass coverslips.
Images were captured with a DFC-350 FX digital camera (Leica, Germany) attached to the fluorescent microscope, and randomly selected areas were analyzed to generate optical-density plots using the Histogram tool in the Image menu of Adobe Photoshop (Adobe Systems, Inc.). The average fluorescent intensities within the selected areas were recorded as arbitrary units, and the relative fluorescent intensities protein (monomeric Kusabira-Orange 2 (mKO2)) were calculated to evaluate the frequency of G1-arrest in fifth-day cetuximab treatment cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from freshly obtained blood (AIH or HC) by Ficoll (GE Healthcare Life Sciences, Piscataway, NJ, USA) gradient centrifugation. Then PBMCs were stained with anti-HLA-DR-PercpCy5.5, anti-CD11b-PE-Cy7, and anti-CD33-BV421 (BD Pharmingen). Moreover, anti-gp130-APC (Biolegend) and anti-IL27RA-Alexa Flour488 (R&D systems) were used and then anti-IL12Rβ2 primary antibody was combined with Alexa Fluor 555-conjugated anti-rabbit IgG secondary antibody (Invitrogen/Life Technologies, UK). The percentages and numbers of HLA-DR^−/lowCD33⁺CD11b⁺ cells (MDSCs) and expression abundance of IL-35 and IL-27 receptors on MDSC were quantified and analyzed by flow cytometry (BD LSRFortessa). For some in vitro experiments, anti-HLA-DR-FITC, and anti-CD11b-APC (BD Pharmingen) were utilized as well. 500,000 events were recorded and analyzed using the FlowJo software version 7.2.2 (Tree Star, Ashland, OR, USA).