Cellular immunofluorescence experiment steps - Database & Sql Blog Articles

**Cellular Immunofluorescence Experiment Steps** **Day 1:** 1. **Washing the cells:** Gently dip the cells that have adhered to the culture plate into PBS three times, each for 3 minutes. This step helps remove any residual media and prepares the cells for fixation. 2. **Fixation:** Fix the cells using 4% paraformaldehyde for 15 minutes. After fixation, rinse the slides with PBS three times, each for 3 minutes to remove any excess fixative. 3. **Permeabilization:** Incubate the slides in 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 minutes. This step allows antibodies to penetrate the cell membrane. Note: If the antigen is located on the cell membrane, this step may be skipped. 4. **Blocking:** After washing with PBS three times (3 minutes each), blot the slides with absorbent paper. Apply a drop of normal goat serum to each slide and incubate at room temperature for 30 minutes to block non-specific binding sites. 5. **Primary Antibody Incubation:** Remove the blocking solution with absorbent paper, without washing. Add an appropriate volume of diluted primary antibody to each slide, cover with a wet chamber, and incubate overnight at 4°C. **Day 2:** 6. **Secondary Antibody Incubation:** Wash the slides with PBST three times (3 minutes each). Blot the slides with absorbent paper, then add a diluted fluorescent secondary antibody. Incubate in a wet chamber at 20–37°C for 1 hour. Afterward, wash the slides again with PBST three times (3 minutes each). *Note: From this point onward, all steps should be performed in a dimly lit or dark environment to prevent photobleaching of the fluorescent signal.* 7. **Nuclear Staining:** Incubate the slides with DAPI in the dark for 5 minutes to stain the nuclei. Then, wash the slides with PBST four times, each for 5 minutes, to remove excess DAPI. 8. **Mounting and Imaging:** Blot the remaining liquid from the slides with absorbent paper. Apply anti-fade mounting medium to seal the coverslip. Examine the samples under a fluorescence microscope and capture images. 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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