Intracellular Protein Staining Kit

Virongy’s Intracellular Protein Staining Kit provides a flexible and reliable tool for immunofluorescent staining of intracellular host and viral proteins, such as host phospho-proteins and viral capsid proteins. Cells are chemically fixed with Fixation Buffer to stop proteolytic enzymes, prevent microbial contamination, and preserve cell morphology. Permeabilization Buffer dissolves fatty acids in the cell membrane to allow antibodies to access intracellular proteins. Staining/Washing Buffer improves immunofluorescent staining of cells by reducing non-specific binding and antigen capping. After staining, cells are ready for fluorescence-activated cell sorting and analysis. Virongy’s intracellular staining kit is compatible with various combinations of antibodies and cytokines to accommodate your individual research needs.

Intracellular staining of p-cofilin in human CD4 T cell CEM-SS

Kit highlights:

• Compatible with a variety of cell types. Single cells can be analyzed quantitatively for multiple parameters at the same time.
• Simple protocol, yielding reliable results. In just three simple steps – fixation, permeabilization, and staining – cells are ready for fluorescence-activated cell sorting and analysis.

Protocol

1. Aliquot approximately 0.5 x 10⁶ cells in 0.5 ml into a 5-ml staining tube.
2. Fix cells by adding an equal volume (0.5 ml) of Fixation Buffer directly into the cell, mix well, and incubate for 10 min at room temperature.
3. Pellet cells by centrifugation at 2,000 rpm (or 600 x g) for 5 min at room temperature. Discard the supernatant.
4. Permeabilize cells by resuspending in 1 ml ice-cold Permeabilization Buffer with vigorous vortexing, and incubate on ice for at least 20 min (at this point, cells can be stored in Permeabilization Buffer at -20°C for up to 5 weeks).
5. Wash cells twice with 2 ml Staining/Washing Buffer.
6. Discard the supernatant. Leave about 100 – 150 μl of Staining/Washing Buffer in the tube.
7. Add diluted primary antibody (dilution is made in Staining/Washing Buffer).
8. Incubate at room temperature for 60 minutes.
9. Wash cells 3 times with 2 ml Staining/Washing Buffer. Pellet cells by centrifugation at 2,000 rpm (or 600 x g) for 5 min at room temperature.
10. Discard the supernatant. Leave about 100 – 150 μl of Staining/Washing Buffer in the tube.
11. Add one diluted secondary antibody to the tube (dilution is made in Staining/Washing Buffer).
12. Incubate at room temperature for 30 minutes in the dark.
13. Wash cells 3 times with 2 ml Staining/Washing Buffer.
14. Resuspend cells in 500 ul Staining/Washing Buffer and analyze by FACS.

This Kit Includes

• Fixation Buffer (250 ml)
• Permeabilization Buffer (250 ml)
• Staining/Washing Buffer (250 ml)