Cellular immunofluorescence experiment steps - Database & Sql Blog Articles

**Cellular Immunofluorescence Experiment Protocol** **Day 1:** 1. **Washing the Cells:** Gently dip the cells that have adhered to the culture plate in PBS (Phosphate Buffered Saline) three times, each for 3 minutes. This step helps remove any residual media and ensures a clean surface for fixation. 2. **Fixation:** Fix the cells using 4% paraformaldehyde for 15 minutes. Afterward, wash the slides three times with PBS, each for 3 minutes, to remove excess fixative. 3. **Permeabilization (if needed):** If you're targeting membrane-bound antigens, incubate the slides in 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 minutes. This allows the antibodies to penetrate the cell membrane. 4. **Blocking:** Wash the slides again with PBS three times, 3 minutes each. Then, blot the slides with absorbent paper to remove excess liquid. Apply normal goat serum to block non-specific binding sites and let it sit at room temperature for 30 minutes. 5. **Primary Antibody Incubation:** Remove the blocking solution with absorbent paper without washing. Add an appropriate dilution of the primary antibody to each slide, ensuring full coverage. Place the slides in a humidified chamber and incubate overnight at 4°C. **Day 2:** 6. **Secondary Antibody Incubation:** Wash the slides with PBST (PBS + 0.05% Tween 20) three times, 3 minutes each. Blot the slides with absorbent paper, then add the diluted fluorescent secondary antibody. Incubate in a wet box at 20–37°C for 1 hour. Wash the slides again with PBST three times, 3 minutes each. *Note: From this point onward, all steps should be performed in low-light or dark conditions to prevent photobleaching of the fluorescent signals.* 7. **Nuclear Staining (DAPI):** Incubate the slides with DAPI (4',6-diamidino-2-phenylindole) in the dark for 5 minutes to stain the nuclei. Afterward, wash the slides with PBST four times, 5 minutes each, to remove excess DAPI. 8. **Mounting and Imaging:** Blot the slides with absorbent paper to remove excess liquid. Apply an anti-fade mounting medium to preserve fluorescence and seal the coverslip. Finally, visualize the cells under a fluorescence microscope and capture images for analysis. This detailed protocol ensures accurate and reproducible results in cellular immunofluorescence experiments. Always follow safety guidelines when handling chemicals like paraformaldehyde and fluorescent dyes.

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