Published in
BioTechniques Protocol Guide 2008
(p.9)
A key to successful identification of biomolecules by immunofluorescence (IF) staining involves careful selection of specific antibodies. Many of R&D Systems antibodies have been extensively tested for IHC/IF applications and are selected based on a high signal-to-noise ratio. The following protocol is intended to be a starting point for intracellular IF staining of rat cortical stem cells. For alternative protocols for cell-surface staining and IHC, please visit R&D Systems website at Web site (external).
Rat cortical stem cells (R&D Systems Catalog # NSC001) were grown on sterile cover slips in 24-well plates according to the protocol outlined in R&D Systems Mouse/Rat Neural Stem Cell Functional Identification Kit (R&D Systems Catalog # SC013).
Remove media and wash cells twice with 1 mL of PBS.
Fix cells with 0.5 mL of 4% paraformaldehyde for 20 minutes at room temperature.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
Permeabilize and block cells with 0.5 mL of PBS containing 10% normal donkey serum, 0.1% Triton X-100, and 1% BSA (Blocking Buffer) for 45 minutes at room temperature.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
Incubate with 300 μl of primary antibody/antibodies diluted in Blocking Buffer overnight at 4°C.Note: A negative control should be performed using Blocking Buffer with no primary antibody.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
Incubate with 300 μl of appropriate R&D Systems NorthernLights™ fluorescent secondary antibody diluted in Blocking Buffer in the dark at room temperature for one hour.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
If desired, counter-stain with DAPI or other counter-stain of choice.
Wash cells three times with 0.5 mL of 0.1% BSA in PBS for 5 minutes each.
Wash the cells once with 0.5 mL of PBS for 5 minutes.
Aspirate PBS from the cover slips and add distilled water. Carefully remove the cover slips with forceps and mount cell side down onto a drop of mounting media (R&D Systems, Catalog # CTS011) on a glass slide.
The slides are ready for microscopic observation.
(Click to enlarge)
