Anti rabbit igg alexa fluor 555

PAMs or Marc-145 cells that were grown in 12-well cell plates or in 15 mm covered, glass-bottomed petri dishes were washed with PBS and were fixed with 4% paraformaldehyde (Beyotime, P0099) for 10 min, and they were then permeabilized with 0.5% Triton X-100 (Beyotime, P0096) in PBS for 5 min. The cells were blocked in Immunol staining blocking buffer (Beyotime, P0260) for 30 min at room temperature. Subsequently, the primary antibodies were incubated overnight at 4°C. After washing to remove unbound antibodies, the cells were incubated with the appropriate Alexa Fluor 488 or 555 conjugated secondary antibodies at room temperature for 1 h in the dark. Washing three times again, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, C0065) for 5 min. Finally, the fluorescence images were taken using an inverted fluorescence microscope (NIKON ECLIPSE Ti2-U) or a confocal laser scanning microscope (TCS-SP5, LEICA). Antibodies used in the immunofluorescence and confocal microscopy: anti-Myc antibody (1:800; Cell Signaling Technology, 2276s), anti-PRRSV N antibody (4A5) (1:1500; Jeno Biotech, 9041), anti-clathrin heavy chain (1:1000, Abcam, ab21679), anti-Mouse IgG Alexa Fluor 488 (1:1000, Cell Signaling Technology, 4408s), and anti-Rabbit IgG Alexa Fluor 555 (1:1000, Cell Signaling Technology, 4413s).

The eyes were harvested on the 7th day after alkali injury and embedded in optimal cutting temperature (OCT) compound after the fixation with 4% paraformaldehyde overnight at 4°C. We blocked 5 μm OCT frozen sections with 3% BSA for 1 h at room temperature and incubated the sections overnight at 4°C with a rat anti-F4/80 antibody (Abcam, ab66440, 1:200), rat anti-CD11b antibody (Abcam, ab8878, 1:200) or rabbit anti-CD31 antibody (Abcam, ab28364, 1:100). Detection of bound anti-F4/80, anti-CD11b or anti-CD31 antibodies was performed using anti-rat IgG Alexa Fluor 488 (Cell Signaling Technology, Germany, 1:1000), anti-rat IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000) or anti-rabbit IgG Alexa Fluor 555 (Cell Signaling Technology, Germany, 1:1000). After counterstaining with DAPI (Abcam, ab228549), the sections were mounted with antifade mounting medium (Beyotime, Shanghai, China). The sections were viewed and photographed with fluorescence microscopy (DMI3000 B; Leica, Wetzlar, Germany).

The following reagents were used in this study: HNP1, HNP2 and HNP3 (Peptide Institute, Inc., Japan); TGF-β (BioLegend Inc., CA, USA); Dulbecco’s Modified Eagle’s Medium (Cytiva, Marlborough, MA, USA); Fetal Bovine Serum (Gibco, Grand Island, NY); ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, CA, USA); LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision Inc., CA, USA); RNeasy Mini Kit (QIAGEN Inc., Hilden, Germany); iScript Reverse Transcription Supermix, SsoAdvanced™ Universal Probes Supermix (Bio-Rad Inc., CA, USA); Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., NY, USA); 1X Protease/Phosphatase Inhibitor Cocktail, Rabbit anti-COL1A1 antibody, Mouse anti-Ki-67 antibody, Rabbit anti-β-actin antibody, Mouse anti-rabbit IgG antibody (HRP conjugate), Anti-rabbit IgG Alexa Fluor 555, Anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technology Inc., MA, USA); Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Sciences Inc., MA, USA); Alliance Q9 chemiluminescence imaging system (Uvitec Inc., UK); Tissue-Tek O.C.T.™ Compound (Sakura, Alphenaan den Rijn, Netherlands).

M1 and M2 macrophages in lesions were identified by co-staining for iNOS (Abcam, #ab15323) or mannose receptor (#HM1049, Hycult Biotech), respectively, and macrophage (Accurate Chemical, #AIA31240), as follows: frozen sections were fixed in acetone (15 min, 4°C) followed by incubation with iNOS antibody (1:100 dilution) overnight at 4°C and then anti-rabbit IgG Alexa Fluor-555 (#4413, Cell Signaling) at 1:1,000 dilution for 1 h, or with mannose receptor antibody (1:100 dilution) overnight at 4°C and then anti-rat IgG Alexa Fluor-555 (#4417, Cell Signaling) at 1:1,000 dilution for 1 h. Next, AIA31240 antibody was used at a 1:100 dilution followed by staining with anti-rabbit IgG Alexa Fluor-488 (#A11008, Invitrogen) at 1:1,000 dilution for 1 h. Slides were mounted using the Prolong Gold Antifade Reagent with DAPI (Cell Signaling, #8961). All antibody dilutions were done using the IHC TEK antibody diluent (IW-1,000) from IHC World.

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by 0.1% Triton X-100 to allow cell permeabilization. Cells were then incubated with the following primary antibodies: anti-Ki67 polyclonal antibody (1:300) (Abcam, Inc., Cambridge, UK), anti-LEF1 polyclonal antibody (1:300), anti-TCF4 polyclonal antibody (1:300), anti-MITF monoclonal antibody (1:300) (Santa Cruz Biotecnology), anti-Cyclin D1 monoclonal antibody (1:300) (Zymed Laoratories, Life Tecnologies, Invitrogen) for 1 hour. Primary antibodies were visualized using anti-rabbit IgG Alexa Fluor 488, anti-mouse IgG Alexa Fluor 488, anti-goat IgG Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) or anti-rabbit IgG-AlexaFluor 555 (Cell Signaling Technology, MA, USA). Nuclei were visualized with 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Srl, Milan, Italy). Fluorescence signals were recorded using a CCD camera (Zeiss, Oberkochen, Germany). The percentage of positive cells for Ki67 was evaluated counting for each melanoma cell populations, a total of 500 cells observed in 10 fields and expressed as mean values ± SD.

MIA Paca-2 and PANC-1 cells were grown on 4 well chamber slides. The cells were washed three times with PBS after SP1 siRNA treatment, and then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton ×100 (Sigma) for 10 min. After three time washing with PBS, incubate cells with 1% BSA in PBST (PBS + 0.1% Tween20) for 30 min. Polyclonal rabbit antibodies against human SP1 (1:200, Abcam, Cambridge, UK), F-actin (1:200, Abcam, Cambridge, UK) were applied overnight at 4 °C. Washed cells were incubated with Secondary Alexa Fluor 555 anti-rabbit IgG (1:1000, Cell signaling) and Alexa Fluor 488 anti-mouse IgG (1:1000, Cell signaling) for 1 hour at room temperature, washed again. Cells were mounted in DAPI Staining Solution (Abcam, Cambridge, UK), and analyzed using confocal microscopy (LSM510, Carl Zeiss, Jena Germany).