Immunofluorescence

Immunofluorescence (IF) technology, based on the principle of antigen-antibody reaction, uses fluorescent antibody (antigen) as a probe to detect the corresponding antigen (antibody) in tissues or cells, and forms a fluorescent antigen-antibody complex in tissues or cells. To achieve the purpose of detecting, monitoring, analyzing and even purifying proteins.
Fluorescent probe types used for protein labeling include anthocyanins, Rhodamine, fluorescein, bioproteins such as GFP, and quantum dots. The fluorescent probe can be attached to the target protein by cyanine dye, Rhodamine dye, where fluorescein is labeled by attaching the cysteine, lysine, tyrosine or N-terminal of the target protein.
Depending on the results, it could eventually be possible to use techniques such as fluorescence microscopy to quantify and monitor target proteins, flow cytometry to sort proteins, or even fluorescent live cell imaging with multiple labeled proteins to monitor living cells^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Melt wax: 72℃ for 1-2 h.

2. Dewaxing: xylene for 20 min.

3. Dehydration: anhydrous ethanol for 3 min, 95% ethanol for 3 min, 75% ethanol for 3 min, TBS for 3 min (three times).

4. Antigen repair: immersed in antigen repair buffer, microwave for 4 min, then put to room temperature, TBS for 3 min (three times).

5. Transparent: Triton X-100 20 min.

6. Seal: Seal with sealing liquid for 30 min.

7. Primary antibody incubation: overnight incubation at 4℃.

8. Washing: TBST 5 min (three times).

9. Incubation of secondary antibodies: incubate at room temperature for 2 h.

10. Washing: TBST 5 min (three times), TBS 5 min (once).

11. Sealing tablets: Use sealing tablets containing anti-quenching agents (whether to add DAPI for nuclear staining according to experimental requirements).

1. Regarding transparency, if the slice is relatively thin, it may not penetrate the film. But if you're studying proteins on the surface of the cell membrane, don't penetrate the membrane.

2. In the process of antigen repair, pay attention to prevent excessive evaporation of buffer when microwave oven heating, do not dry. The repair fluid and repair strength are determined according to the tissue.