Alexa fluor 594 igg h l

The marker protein of different macrophage phenotypes was fluorescently labeled by antigen–antibody binding. The surface fluorescence density was tested to evaluate the macrophage phenotype on various samples. Macrophages were seeded on the surface of the samples with a density of 1 × 10⁵ cells per well at 37°C and cultured for 4 days. After washed by PBS, the cells were fixed with 4% paraformaldehyde at 4°C in the dark. After permeabilized for 2 min with 0.1% (v/v) Triton X-100, 1% wt% BSA (Sigma-Aldrich, USA) was added to block the Fc-receptor. Then, the cells were incubated with CD206 (Abcam, UK, 1:50) and inducible nitric oxide synthase (iNOS) (Novus, USA, 1:50) antibody at 4°C overnight. Afterwards, cells were incubated with donkey anti-mouse secondary antibody IgG H&L Alexa Fluor 594 (Abcam, UK, 1:200) and donkey anti-rabbit IgG H&L Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific Inc., USA, 1:200) for 2 h in the dark. After washed with PBS,4',6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei in the dark at room temperature for 10 min. Confocal laser scanning microscope (Leica TCS SP8, Germany) was used for observation, and the fluorescence data were quantitatively analyzed using Image J software.

The spinal cord tissues of the TRANSPLANT+PEG group and the TRANSPLANT group were cut into 10‐μm longitudinal sections, while the bridging tissue of the spinal cord of one beagle in the TRANSPLANT+PEG group was cut into 10‐μm coronal sections. The sections were then mounted onto silanized glass slides. Slides were air‐dried for 15 min and washed in PBS before blocking in 10% normal bovine serum and 0.1% Triton X‐100 in PBS at room temperature. Primary antibodies diluted in Dako antibody diluent were applied overnight in a humidified chamber at 4°C. Primary antibodies included anti‐neurofilament heavy polypeptide (1:100, Abcam) and anti‐glial fibrillary acidic protein (GFAP; 1:1000, Abcam). Slides were then washed and secondary antibodies (IgG‐H&L, Alexa Fluor 594; 1:500, Abcam) that were diluted in PBS were applied for 45 min at room temperature. This procedure was repeated again for a duration of 30 min using IgG‐H&L, Alexa Fluor 488 (1:500, Abcam). Finally, the slides were washed in PBS prior to being mounted with a coverslip using VECTASHIELD (Vector Laboratories).

Bacterial cultures were grown and induced as previously described [34 (link)]. Cells from approximately 0.5 mL of culture were harvested once with PBS, washed four times with PBS, and resuspended in 1 mL PBS without bovine serum albumin (BSA) at 4 °C. The resulting bacterial suspensions were immediately analysed by flow cytometry using a MACSQuant analyser (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's instructions.
For indirect immunofluorescence microscopy, the stained cells were resuspended in 100 µL of PBS and stained with monoclonal anti-c-Myc antibody and goat anti-mouse Alexa Fluor^® 594 (IgG H&L) (Abcam; Cambridge, UK) and visualised under an Axio Observer.z1 microscope (Zeiss, Oberkochen, Germany) using excitation and emission wavelengths of 591 and 614 nm, respectively, and a bright field photomultiplier tube for transmitted light.