Cytoplasmic microtubule display assay
Cytoplasmic microtubule display assay
Microtubules are a common structure in eukaryotic cells. They are hollow tubular fibers that are polymerized from heterodimers of a and beta microtubule proteins and a few microtubule-binding proteins. In different types of cells, microtubules have the same basic morphology. The single tubules in microtubules are distributed in the cytoplasm in the form of a network or bundles; the duplex tubules constitute the peripheral part of cilia and flagella; and the triplet tubules constitute the centrioles as well as the cilia and flagellar basal bodies. Microtubules can be observed by electron microscopy and immunocytochemical techniques, the more commonly used of which are indirect immunofluorescence.
Principle
The basic principle of the indirect immunofluorescence technique to show cytoplasmic microtubules is to first use anti-tubulin immune serum (primary antibody) to incubate with in vitro cultured cells, which binds specifically to intracellular microtubules, and then use fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit (IgG) serum (secondary antibody) to bind to the primary antibody by incubation, which indirectly labels the microtubules with fluorescein. Under a fluorescence microscope, a network of microtubules stretching within the cytoplasm can be seen. In addition to its high sensitivity, the indirect immunofluorescence method requires the preparation of only one kind of indirect fluorescent antibody, which can be applied to the labeling display of a variety of primary antibodies, and is widely used in the structural localization and morphological display of biological macromolecules.
Operation method
Indirect immunofluorescence reveals cytoplasmic microtubules
Principle
The basic principle of the indirect immunofluorescence technique to show cytoplasmic microtubules is to first use anti-tubulin immune serum (primary antibody) to incubate with in vitro cultured cells, which binds specifically to intracellular microtubules, and then use fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit (IgG) serum (secondary antibody) to bind to the primary antibody by incubation, which indirectly labels the microtubules with fluorescein. Under a fluorescence microscope, a network of microtubules stretching within the cytoplasm can be seen. In addition to its high sensitivity, the indirect immunofluorescence method requires the preparation of only one kind of indirect fluorescent antibody, which can be applied to the labeling display of a variety of primary antibodies, and is widely used in the structural localization and morphological display of biological macromolecules.
Materials and Instruments
Equipment: Move The basic procedure for the demonstration of cytoplasmic microtubules by indirect immunofluorescence can be divided into the following steps: After drying slightly, add glycerol-PBS (91) dropwise on the slide and cover with a slide. Caveat 1 Wash each step well and absorb the water (but do not dry it out) so as not to dilute the antibodies or reagents in the next step, so that a clear fluorescent image can be obtained.2 Appropriate antibody dilution. Antibody dilution mainly refers to the "primary antibody", because the "primary antibody" in the specific antibody concentration is the key. The "primary antibody" and "secondary antibody" should be tested for the optimal dilution before use, with the strongest fluorescence of the specific staining reaction and negative non-specific staining as the best.3 Incubation time is 30-60 min, and the temperature is usually 37 ℃, which can enhance the antigen-antibody reaction, but it should be carried out in a wet box to prevent the specimen from drying out and causing failure.4 The specimen should be observed immediately after staining, and the fluorescence will gradually diminish after too long a period of time. If the specimen is stored in a polyethylene bag at 4 ℃, it can delay the time of fluorescence weakening and prevent the evaporation of the sealant. For more product details, please visit Aladdin Scientific website.
Cell culture equipment, inverted microscope, fluorescence microscope, refrigerator, micro-sampler (100 μl), oscillator, aluminum box, routine laboratory instruments, in vitro cultured fibroblasts.
Reagents:
① 0.01 mol/L (pH 7.2) phosphate buffered saline (PBS)
② PEMP buffer
① 0.01 mol/L (pH 7.2) phosphate-buffered saline (PBS) ② PEMP buffer ③ Fixation solution (3.7% formaldehyde-PEMD solution)
④ 0.5% Triton X-100/PEMP solution
⑤ 1% Triton X-100/PBS solution ⑥ Rabbit anti-microtubulin antibodies
⑥ Rabbit anti-microtubule protein antibody
⑦ Fluorescein isothiocyanate (FITC)-sheep anti-rabbit antibody
⑧ Glycerol-PBS buffer 9:1, pH 8.5~9.0)
A Fibroblasts are cultured on slides and removed when they have grown to 10-10% of their length.
B Slides with monolayers of cells are rinsed in PEMP buffer.
C Slides are processed for 1.5-2 min in a pre-warmed 0.5% Triton X-100/PEMP solution at 37 °C.
D Slides are washed twice with PEMP. E 3.7% formaldehyde-PEMD solution is fixed for 30 min at room temperature. F pH 7.2 PBS is washed twice for 5 min each.
E 3.7% formaldehyde-PEMD solution was fixed for 30 min at room temperature.
F PBS pH 7.2 washed twice for 5 min each time. Filter paper was used to blot up the remaining liquid.
G Primary antibody was conjugated. Place the cell side upwards in an aluminum box containing PBS, carefully add 40 μl of rabbit anti-microtubule protein antibody diluted appropriately onto the cell layer, close it tightly, and incubate for 40-60 min at 37 ℃.
H Remove the slide, aspirate off the antibody solution, and put it into a 35 mm small staining cylinder, and then wash it according to the following sequence: PBS → 1% Triton X-100/PBS → PBS, each time for 5-10 min. Wash in the following order: PBS→1% Triton X-100/PBS→PBS, each time for 5~10 min, with agitation or gently shaking on a shaker to wash away unbound antibody. Remaining liquid was removed by filter paper and dried slightly.
I-conjugated secondary antibody. Add 40 μl of diluted FITC-goat anti-rabbit antibody onto the cell surface as in (7).
J Remove the slide, aspirate the remaining antibody, and rinse by immersion to remove unbound antibody. The procedure is the same as (8). Finally, pass through deionized water 2 times
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