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Immunofluorescence Staining of Expanded Primary Cells or Stem Cells Includes the Following Steps: (For the full protocol, refer to the link: https://www.researchgate.net/publication/394048541_Immunofluorescence_IF_Staining_Technology_Protocol_Provided_by_MCE)

1. Cell Fixation

Fix cells at room temperature using 4% paraformaldehyde (PFA) for 10–30 minutes (20 minutes is commonly used). Alternatively, fix with pre-cooled 100% methanol at –20°C for 5 minutes. The goal of fixation is to preserve cellular structures and maintain antigen integrity. After fixation, wash the cells three times with PBS, each for 5 minutes, to remove residual fixative.

2. Cell Permeabilization

If the target protein is located intracellularly, permeabilization is necessary to allow antibody access. Use 0.1%–0.25% Triton X-100 or 0.5% Saponin in PBS and incubate for 10 minutes. After permeabilization, wash the cells three times with PBS, each for 5 minutes.

3. Blocking

Block non-specific binding sites using 1%–5% BSA (e.g., HY-D0842, MCE) or 10% normal goat serum (or the serum from the host species of the secondary antibody), diluted in PBS-T (PBS containing 0.1% Tween 20). Incubate for 30 minutes at room temperature.

4. Primary Antibody Incubation

Dilute the primary antibody in blocking buffer at the recommended dilution. Incubate the cells for 1 hour at room temperature or overnight at 4°C in a humidified chamber or protected from light to ensure optimal antigen–antibody binding. After incubation, wash the cells three times with PBS, each for 5 minutes.

5. Secondary Antibody Incubation

Dilute the fluorescently labeled secondary antibody (species-specific to the primary antibody) in blocking buffer. Incubate at room temperature for 1 hour, protected from light. Following incubation, wash the cells three times with PBS, each for 5 minutes, to remove any unbound antibodies.

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