Microfilament staining and morphology observation experiment
Microfilament staining and morphology observation experiment
The network of fibers crisscrossing the cytoplasm of eukaryotic cells is called the cytoskeleton. According to the fiber diameter, composition and assembly structure are divided into microtubules, microfilaments and intermediate fibers. The current means of observing the cytoskeleton are mainly electron microscopy, indirect immunofluorescence technique, enzyme labeling and histochemistry. Microfilaments are fibers composed of actin. Single microfilaments are about 7 nm in diameter and are not visible under the light microscope. In different kinds of cells, they form different subcellular structures together with certain binding proteins, such as muscle filaments, intestinal epithelial microvillus axons and stress fibers. In this experiment, Coomassie brilliant blue R250 was used to show the stress fibers composed of microfilaments. Stress fibers are particularly well-developed in adherent cells cultured in vitro and are associated with the attachment of cells to the culture matrix and the maintenance of a flat spreading cell shape.
Principle
The basic principle of the microfilament staining and morphology observation experiment is that the Caulmers Brilliant Blue R250 can stain a variety of proteins, and is not specific for staining microfilaments. However, under the conditions of this experiment, the microtubule structure was unstable, and some types of fibers were too fine to be distinguished under the light microscope. Therefore, we see mainly stress fibers composed of microfilaments with a diameter of about 40 nm.
Operation method
Microfilament staining and morphology observation experiment
Principle
The basic principle of the microfilament staining and morphology observation experiment is that the Caulmers Brilliant Blue R250 can stain a variety of proteins, and is not specific for staining microfilaments. However, under the conditions of this experiment, the microtubule structure was unstable, and some types of fibers were too fine to be distinguished under the light microscope. Therefore, we see mainly stress fibers composed of microfilaments with a diameter of about 40 nm.
Materials and Instruments
Equipments: cell culture equipment, inverted microscope, thermostat, microscope, 25 ml weighing flasks, routine laboratory instruments, in vitro cultured adherent growth cells, onion bulbs. Move The basic process of microfilament staining and morphology observation can be divided into the following steps: Caveat 1 Add the reagents slowly along the inside of the weighing flask to avoid dripping directly onto the coverslip; wash the cells gently to avoid dislodging.2 Extraction, fixation and staining should be performed in a covered weighing flask with the cell side of the coverslip always facing up.3 Extraction of miscellaneous proteins and lipids with 1% Triton X-100 should be done as a pre-test, long extraction time will destroy the cell structure, and short extraction time will cause large background interference.4 Stress fiber is a dynamic structure, when the cell is fully adhered to the wall and spread the fiber is erect and abundant; on the contrary, when the cell shrinks and becomes rounded, the stress fiber will be bent, and even partially depolymerized and disappeared, which is scarce. For more product details, please visit Aladdin Scientific website.
Reagents:
① 6 mmol/L PBS (pH 6.5)
(Liquid A: NaH2PO4-2H2O, 936 mg/1000 ml. Liquid B: Na2HPO4-12 H2O, 2148 mg/1000 ml.) Working solution: Liquid A 68.5 ml + Liquid B 31.5 ml (with NaHCO
3
Working solution: liquid A 68.5 ml + liquid B 31.5 ml (adjust pH to 6.5 with NaHCO 3)
② M buffer (pH 7.2)
(Imidazole, 3.40 g; KCl, 3.71 g; MgCl2-6H2O, 101.65 mg; EGTA (ethylene glycol diethyl ether tetraacetic acid), 380.35 mg; EDTA (ethylenediaminetetraacetic acid), 29.22 mg; mercaptoethanol (mer-captoethanol), 0.07 ml; glycerol, 292 ml; add distilled water to 1000 ml (pH adjusted to 7.2 with 1 mol/LHCl).
③ 1% Triton X-100
③ 1% Triton X-100 ④ 0.2% Kaomas Brilliant Blue R250 dye solution
⑤ 3% glutaraldehyde
(I) Display and observation of microfilaments in animal cells
A Collection of materials Cells were cultured on coverslips (a corner was cut off in order to differentiate between front and back sides of the cells), and were removed when the growth density reached 10-10-10, and placed in a weighing flask with the cells facing upward, and washed 3 times with 6 mmol/L PBS for 1 min each time.
B Extraction Discard the PBS washings and put 2 ml 1% Triton X-100 into a weighing flask, cover it with the lid, and put it into an aluminum box with a wet gauze. Remove the PBS wash solution, add 2 ml of 1% Triton X-100, close the lid of the weighing flask, place it in an aluminum box lined with wet gauze, and place it in a 37℃ thermostat for 25-30 min.
C Rinsing Discard the 1% Triton X-100, and add 2 ml of M buffer to gently wash the cells for 3 times, each time for 2 min. M buffer has the effect of stabilizing the cytoskeleton.
D Fixation Dry the cells briefly, and add 2 ml of 3% glutaraldehyde, and fix the cells for 15 min. After drying, add 2 ml of 3% glutaraldehyde and fix the cells for 15 min.
E Rinse Discard the fixative and wash the cells gently with 6 mmol/L PBS 3 times for 2 min each time.
F Staining Discard the washings, stand the small cover slip on absorbent filter paper, and absorb the water at the edge of the specimen. Add 2 ml of 0.2% Kaomas Brilliant Blue R250 staining for 20 min.
G Rinsing Wash the specimen gently with distilled water to remove the staining solution, blot the edges of the specimen with absorbent filter paper, air-dry it, and then visualize it directly or seal it with resin.
(II) Display and observation of microfilaments in plant cells
A Tear the inner epidermis of onion bulb with tweezers, about 1 c㎡ in size, and put it into a weighing bottle containing 6 mmol/L PBS to make it sink, and process it for 5-10 min.
B Extraction Abandon the PBS, add 2 ml of 1% Triton X-100, put the lid of the weighing bottle, put it in an aluminum box with a wet gauze, and process it for 30 min in a constant temperature box at 37℃. C Rinse and rinse the specimen. Place in an aluminum box lined with wet gauze and place in a 37℃ incubator for 30 min.
C Rinse Discard 1 % Triton X-100 and add 2 ml M buffer to gently wash the inner epidermis of the onion, each time for 3-5 min for a total of 3 times.
D Fixation Add 2 ml 3% glutaraldehyde and fix for 20 min.
E Rinse Discard the fixation solution and add 2 ml 6 mmol/L PBS to gently wash the inner epidermis of the onion, each time for 3-5 min for a total of 3 times, and remove the residual solution with an absorbent filter. F Chromatographic filter paper Absorb the residual liquid.
F Staining Add 2 ml of 0.2% Kaomas Brilliant Blue R250 staining for 20 min.
G Preparation Discard the staining solution and wash gently with distilled water for 3 times. Lay the specimen flat on a slide and cover the slide. Microscopic examination.