Alexa fluor 488 goat anti mouse igg h l antibody

Immunofluorescence microscopy was performed according to the method reported previously (Kobori et al., 1992 (link)). P. pastoris was induced for 144 h in BMMY, and then were harvested by centrifugation for 1 min at 10,000 rpm and 4°C. The supernatant was discarded. Harvested cells were washed with distilled water and resuspended in ice-cold phosphate-buffered saline (PBS, pH 7.4), with 10 mg/mL of bovine serum albumin to block the cell surface. Anti-flag monoclonal antibody (Agilent, United States) was used as the primary antibody. Cell suspension was incubated with the primary antibody at a dilution of 1:200 in a total volume of 200 mL at room temperature for 2 h. Next, cells were washed twice with PBS and exposed to the secondary Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody (Invitrogen, United States) at a final concentration of 10 ng/mL for 1 h at room temperature. Cells were washed three times with PBS and analyzed by using the BX51 fluorescence microscopy (Olympus, Japan). GS115/pPIC9K was also processed in the same procedure to serve as negative controls.

Relative PrP surface expression levels of PK1 clones were determined by FACS. Briefly, 1 × 10⁶ cells were pelleted at 300 × g for 4 min and washed with PBS. Cell pellets were then fixed on ice with 4% paraformaldehyde/PBS for 30 min. After washing with PBS, cells were incubated with 5 μg of ICSM 18 in PBS/0.1% bovine serum albumin (BSA) for 30 min. Cells were washed again with PBS/0.1% BSA, spun, and incubated with a 1:200 dilution of Alexa Fluor 488-goat anti-mouse IgG (H+L) antibody (Invitrogen, Paisley, UK) in PBS/0.1% BSA for 30 min. After washing, PrP surface expression levels were determined using a FACS Calibur flow cytometer (BD Biosciences). Background levels of fluorescence were determined by labelling cells with secondary antibody only.

RAW264.7 cells were placed on 96-well plate (5 × 10³ cells/well) and incubated for 72 h. For actin cytoskeleton and focal adhesion staining, cells were washed twice with PBS, fixed for 20 min with 4% paraformaldehyde in PBS (PH 7.4) at normal temperature. Cells were then permeabilized in PBS containing 0.1% Triton X-100 for 5 min at room temperature, and then washed twice using PBS. Then, cells were washed by blocking buffer (1% BSA in PBS for 30 min). Cells were incubated for 1 h at room temperature with a primary antibody (Anti-Vinculin) diluted in blocking solution to a working concentration (1:300). This was followed by washing three times (5–10 min each) with washing buffer (0.05% Tween-20 in PBS). Cells were labeled for 1 h at room temperature with a secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H + L) Antibody, Invitrogen, Carlsbad, CA, USA) (1:500), and TRITC conjugated Phalloidin (1:500) was diluted in PBS. Cells were then washed three times as above. Nuclei counter staining was performed by incubating cells with DAPI (1:1000) for 5 min at room temperature, followed by washing cells for three times with washing buffer and then mounted for fluorescence microscopy as previously described.

Cells were incubated in 96-well plate (5 × 10³ cells/well) and induced with M-CSF (50 ng/ml) and RANKL (100 ng/ml). Procedures were described in previous study10 (link). In brief, cells were washed and fixed for permeabilization. After blocking, primary antibody (Anti-Vinculin) was then diluted to a working concentration (1:300) in blocking solution, and cells were incubated for 1 hour at room temperature. Secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H + L) Antibody, Invitrogen) ((1: 500) and TRITC conjugated Phalloidin (1: 500) was diluted in 1 × PBS and cells were incubated for 1 h at room temperature. Nuclei counterstaining was performed by DAPI (1: 1000) for 5 minutes followed by fluorescence microscopy and confocal microscopy observation.

Bone marrow macrophages (BMMs) were separated and cultured with M-CSF (50 ng/ml) for 24 h to obtain BMMs. Cells were cultured in α-minimal essential medium (MEM) containing 10% FBS and 1% Penicillin-streptomycin solution. For tartrate resistant acid phosphatase (TRAP) stain, cells were cultured in a 96-well plate at a density of 5 × 10³ cells/well with different stimulations. Cells were fixed in 4% paraformaldehyde for 20 min and then stained with TRAP staining solution (0.1 mg/ml of naphthol AS-MX phosphate, 0.3 mg/ml of Fast Red Violet LB stain) according to the manufacturers’ instructions. Relative TRAP activity was measured by colorimetric analysis. For immunofluorescent stain, cells were cultured on glass sheet in a 12-well plate at a density of 4 × 10⁴ cells/well with different stimulations. In brief, on day 4, cells were washed and fixed for permeabilization. After blocking, primary antibody (Anti-Vinculin) was then diluted to a working concentration (1:300) in blocking solution, and cells were incubated for 1 h at room temperature. Secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H + L) Antibody, Invitrogen) (1:500) and TRITC conjugated Phalloidin (1:500) was diluted in 1 × PBS and cells were incubated for 1 h at room temperature. Nuclei counterstaining was performed by DAPI (1:1000) for 5 minutes followed by fluorescence microscopy.

Samples were cultured for 7 days, fixed in 4% paraformaldehyde at room temperature for 20 min, and rinsed with PBS before permeabilization by 0.3% Triton X-100 (Sigma, USA) in PBS for 15 min on ice. After being rinsed with PBS, samples were blocked by 2.5% BSA (Sigma, USA) in PBS at room temperature for 60 min. The cell sheets were incubated with Cadherin primary antibody (ab6528, 1 : 100, Abcam, UK) in 1% BSA at 4°C overnight after rinsing three times with PBS. Cell sheets were incubated with a secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) antibody, 1 : 400, Invitrogen, USA) at room temperature and kept in the dark for 60 min. Cell nuclei were assessed by Hoechst 33342 as mentioned above. Samples were observed by laser scanning confocal microscopy (IX81, Olympus, Japan). Images and figures were analyzed by Image-Pro 6.0 software (Media Cybernetics, USA).

Transfected MIN6 and islet cells in chamber slides were fixed by 4% paraformaldehyde and stained with primary rabbit polyclonal insulin antibody (1:500 dilution, Santa Cruz Biotech) or a mouse monoclonal insulin antibody (1:500 dilution, Sigma) and secondary antibodies Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody or Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody (1:1000 dilution, Invitrogen). Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) was used for nuclear staining. Images were captured with ZEN Imaging Software (ZEISS, Thornwood, NY) using constant exposure parameters for each fluorescence channel.