Alexa fluor 647 conjugated antibody

Cells were grown on sterile glass coverslips, treated with different conditions, and then washed twice with PBS. For the observation of neurite outgrowth, the cells were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 15 min and then permeabilized with 0.1% Triton X-100 (Bioshop Canada Inc., Burlington, Ontario, Canada) in PBS for 15 min at 37°C. Then, 1% bovine serum albumin (BSA) was used for blocking the non-specific protein binding. The cells were then incubated overnight with primary anti-beta-III tubulin (1:200 dilution; Gentex, Irvine, CA, USA) at 4°C. After being rinsed with PBS, NSC-34 cells were incubated with a secondary Alexa Fluor 647-conjugated antibody (1:1000 dilution; Abcam, Cambridge, MA, USA). Finally, coverslips were mounted in 10 μl glycerol-based mounting medium.
For the observation of nuclear morphology, the cells were fixed in 4% PFA (Sigma-Aldrich) for 15 min, and then the coverslips were directly mounted in 10 μl mounting medium containing DAPI (Abcam). All the fluorescent images were observed and recorded using Zeiss Axio Imager A1 microscope.

BMDMs or BMDMs with GFP-transfected were stained with PE-labeled anti-F4/80 (eBioscience, San Diego, CA, USA) or FITC-labeled anti-CD11b (eBioscience, San Diego, CA, USA). Prepared HNPCs were resuspended in PBS at 4 °C and pre-incubated with TruStain FcX™ (anti-mouse CD16/32) antibody (BioLegend Way, San Diego, CA, USA) to minimize non-specific antibody binding, then HNPCs were stained with PE-labeled anti-F4/80 antibody (eBioscience, San Diego, CA, US), FITC-labeled anti-CD11b antibody (eBioscience, San Diego, CA, US), and anti-GFP antibody (Abcam, Cambridge, MA, USA) with secondary antibody Alexa Fluor^® 647-conjugated antibody (Abcam, Cambridge, MA, USA). FACS analysis was performed using a FACS Calibur^TM flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA). Data were analyzed with the FlowJo software version 10.0 (FlowJo, Ashland, OR, USA).