Immunocytochemistry and Immunofluorescence for HA-Tag Staining Protocol

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Immunocytochemistry and immunofluorescence for HA-tag staining protocol Requirements: 1. Coverslips to grow cells (Fisherbrand coverglass Cat# 12-545-83) 2. 12 well cell culture plates (Corning Incorporated Cat # 3513 or Fisher cat# 07-200-82) 3. Paraformaldehyde (Sigma Cat# P6148) 4. Image-IT Fx Signal enhancer (Invitrogen# 136933) 5. Normal goat serum (Jackson ImmunoResearch- 005-000-121) 6. Primary antibody 7. Secondary antibody conjugated with fluorescent dye 8. Mounting medium with DAPI (Vectashield mounting medium for fluorescence with DAPI. Vector Laboratories Cat # H-1200) 9. Slides ( Fisher brand Cat# 12-550-17) Procedure: 1. Sterilize Circular cover slips by dipping them in absolute ethyl alcohol and flaming 2. Place each coverslip into each well of 12-well culture plate and leave it under UV light for 30 min prior to cell seeding 3. Trypsinize the cells and count under the microscope using hemocytometer. Seed 1×105 cells (1 ml culture medium) in each well containing cover slip 4. Allow cells to attach on to the cover slip at 37 °C in presence of 5% CO2 for 24 h 5. Take the attached cells for immunostaining: Prefix the cells attached on the surface of the cover slips for 5 min by adding 500 ul of 4% paraformaldehyde into the culture medium (1ml) 6. Remove the prefixing medium and fix the cells with 4% paraformaldehyde for 15 min 7. Wash the paraformaldehyde fixed cells with PBS for 3 times 10 minutes each 8. Permeabilize the fixed cells with PBST (PBS+0.25% Triton X-100) for 5 min 9. Wash the cells with PBS for 3 times 10 minutes each 10. Apply 3-4 drop of Image-IT TM FX signal enhancer (sufficient volume to cover the surface of the cover slip) and incubate for 30 min at room temperature in humid environment 11. Wash the cells with PBS for 3 times 10 minutes each 12. Incubate the cells with blocking buffer (PBS with 5% normal goat serum) at 4°C overnight to block unspecific binding (This step can be performed at room temperature for 1 h) 13. Wash the cells with PBS for 3 times 10 minutes each 14. Primary antibody: Incubate the cells with primary antibody (PBS with 1% BSA + primary antibody) at 4°C overnight (This step can be performed at room temperature for 1 h) 15. Wash the cells with PBS for 3 times 10 minutes each 16. Secondary antibody: Incubate the cells with Secondary antibody (PBS with 1% normal goat serum + Secondary antibody) at 4°C overnight (This step can be performed at room temperature for 1 h) 17. Wash the cells with PBS for 3 times 10 minutes each 18. Mount the cover slip in a glass slide using a mounting medium with DAPI (nuclear stain) 19. Take the pictures under fluorescent microscope 20. Apply nail polish at the edge of the cover slip and store the slides at 4°C till the nail polish dries. Then store the slides at -20°C NOTE: - Add enough volume of all the solutions to cover the cover slip (500 ul) - The procedure was carried out at RT unless otherwise specified .

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