Introduction of integrated cellular immunofluorescence technology

Immunofluorescence technology (Immunofluorescence technique), also known as fluorescent antibody technology, is the earliest development of labeling immunotechnology. It is a technology established on the basis of immunology, biochemistry and microscope technology. For a long time, some scholars have tried to combine antibody molecules with some tracer substances, and use antigen-antibody reactions to locate antigen substances in tissues or cells.

One. Traditional immunofluorescence experimental procedures

(1) Experimental materials

Cell sample

2. Reagents and kits: Poly A Quan; 70% methanol; C Tong; PBS; Triton; BSA; DAPI dye solution; DABCO; Tris; glycerin

3. Instruments and consumables: slides

(2) Experimental steps

Cell slide immunofluorescence experimental steps

first day:

1. Dip the slides of the climbed cells in the culture plate with PBS for 3 times, 3 min each time;

2. Fix the slide with 4% Poly-Quan for 15 min, and rinse the slide 3 times with PBS for 3 min each time;

3. 0.5% Triton X-100 (prepared in PBS) permeable at room temperature for 20 min (the antigen expressed on the cell membrane is omitted in this step);

4. Dip the slides in PBS for 3 times, each time for 3 minutes, absorb the PBS with absorbent paper, add normal goat serum onto the slides, and block at room temperature for 30 minutes;

5. Absorb the blocking solution with absorbent paper, do not wash, add enough diluted primary antibody dropwise to each slide and put it into the wet box, incubate at 4 ℃ overnight;

the next day:

6. Add fluorescent secondary antibody: wash the slides 3 times with PBST for 3 min each time, absorb excess liquid on the slides with absorbent paper and add diluted fluorescent secondary antibody dropwise, incubate in a wet box at 20-37 ℃ for 1 h Dip the slices 3 times, 3 min each time; Note: Starting with the addition of the fluorescent secondary antibody, all subsequent steps should be performed in a dark place.

7. Counterstaining nuclei: add DAPI dropwise and incubate for 5 min in the dark to stain the specimen, and wash off excess DAPI with PBST for 5 min × 4 times;

8. Absorb the liquid on the slides with absorbent paper, cover with a mounting solution containing an anti-fluorescence quencher, and then observe and collect the image under a fluorescent microscope

Two main problems of traditional immunofluorescence experiments


1. Climbing effect is not good

2. Large amount of cells and reagents, high cost

3. Uneven distribution of dye liquor

4. Cumbersome operation, time-consuming and laborious


Technical characteristics:

1. Quick and easy sample preparation, the integrated culture room simplifies the immunofluorescence staining scheme

2. Uniform cell distribution, the geometry of the channel slide can produce a uniform cell distribution

3. Affordable experimental protocol, requiring a small amount of cells and reagents

4. High-resolution imaging, suitable for wide-field fluorescence, confocal imaging, FRAP-FRET-FLIM and high-quality phase contrast imaging (Phase Contrast)


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