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

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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

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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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