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        Immunocytochemistry Protocol Using Anti-GFP Antibodies(使用抗GFP抗体的免疫细胞化学实验方案)

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        1710

        实验试剂

         

        X Dulbecco’s Phosphate-buffered saline (D-PBS)

        Fixative solution: 4% Formaldehyde solution in PBS, pH 7.4

        Permeabilizing solution: 0.25% Triton® X-100 in PBS, pH 7.4

        Blocking solution: 5% Normal Goat serum in PBS, pH 7.4

        Conjugated secondary anti-chicken IgG antibody for detection

        1X Phosphate-buffered saline (PBS) pH 7.4

        实验步骤

         

        1.        Remove the media from the cells grown on cover slips. Rinse the cells twice for 1 minute each in D-PBS.

        2.        Fix the cells in Fixative solution (4% formaldehyde in PBS) for 30 minutes at room temperature with gentle agitation in the dark. Remove the solution.

        3.        Wash the cells twice in PBS for 1 minute each with gentle agitation. Remove the PBS.

        4.        Permeabilize the specimen with Permeabilization solution (0.25% Triton® X-100 in PBS) for 5 minutes at room temperature with gentle agitation in the dark. Remove the solution.

        5.        Wash the cells twice in PBS for 1 minute each with gentle agitation. Remove the PBS.

        6.        Add Blocking solution (5% Normal Goat Serum in PBS, pH 7.4) to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

        7.        Wash the cells twice in PBS for 1 minute each with gentle agitation.

        8.        Prepare a 1:400 dilution of anti-GFP chicken antibody in PBS to obtain a final antibody concentration of 5.0 µg/mL.

        9.        Remove the PBS and add the diluted primary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

        10.    Wash the cells twice in PBS for 1 minute each with gentle agitation.

        11.    Prepare the appropriate conjugated secondary antibody in PBS according to the manufacturer’s recommendations.

        12.    Remove the PBS and add the diluted secondary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution.

        13.    Wash the cells twice in PBS for 2 minutes each with gentle agitation. After the final wash, add PBS to the sample. The sample is now ready for imaging and detection using an appropriate method of choice.

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