Alexafluor647 goat anti rabbit secondary

HEK293A or NG2-OSBP cells were seeded at approximately 50% confluence on 20 µg/mL entactin-collagen-laminin coated 8-well glass slides (MP biomedicals ICN6040805) and returned to the incubator. After 1 hour, they were fixed adding formaldehye (Fisher Scientific 50-980-487) to the medium to reach 4% final concentration (concentrated formaldehyde prepared in water and 10× PBS to make an isotonic solution). After 15 minutes at room temperature, wells were rinse 3 times with 50 mM NH₄Cl in PBS to quench unreacted aldehyde, then permeabilized with 0.2% triton X-100 in PBS for 5 minutes. Next, they were rinsed three times in PBS and blocked for 30 minutes in blocking solution (PBS + 5% by volume normal goat serum, ThermoFisher PCN5000). Staining was for 1 hour with 1:500 anti-OSBP (Atlas Antibodies HPA039227) followed by 30 minutes with 1:200 AlexaFluor647 goat anti-rabbit secondary (ThermoFisher A-21245) in blocking solution. Cells were rinsed 4 times in PBS, once in MilliQ water and mounted in ProLong Diamond (ThermoFisher P36961) and #1.5 coverglass. Images were acquired by confocal microscopy.

HEK293A or NG2-OSBP cells were seeded at approximately 50% confluence on 20 μg/ml entactin-collagen-laminin coated 8-well glass slides (MP biomedicals ICN6040805) and returned to the incubator. After one hour, they were fixed adding formaldehye (Fisher Scientific 50–980-487) to the medium to reach 4% final concentration (concentrated formaldehyde prepared in water and 10x PBS to make an isotonic solution). After 15 minutes at room temperature, wells were rinse 3 times with 50 mM NH₄Cl in PBS to quench unreacted aldehyde, then permeabilized with 0.2% triton X-100 in PBS for 5 minutes. Next, they were rinsed three times in PBS and blocked for 30 minutes in blocking solution (PBS + 5% by volume normal goat serum, ThermoFisher PCN5000). Staining was for 1 hour with 1:500 anti-OSBP (Atlas Antibodies HPA039227) followed by 30 min with 1:200 AlexaFluor647 goat anti-rabbit secondary (ThermoFisher A-21245) in blocking solution. Cells were rinsed 4 times in PBS, once in miliQ water and mounted in ProLong Diamond (ThermoFisher P36961) and #1.5 coverglass. Images were acquired by confocal microscopy.