Experimental principle
The cells are interwoven by microfilaments, microtubules, intermediate fibers, etc. to form a very complex three-dimensional network structure. They play an important role in the maintenance of cell shape, intracellular material transport, cell movement, and the relative position of various structures within the cell, so they are called cytoskeleton.
The cytoskeleton is unstable under generally fixed conditions, such as low temperature, high pressure, acid treatment, and the like. When the cells are washed by appropriate means, such as M-buffer, the stability of the cytoskeleton can be improved. The fixation of glutaraldehyde at room temperature can better preserve the components of the cytoskeleton. In addition, the Triton X-100 treatment can extract a part of the cytoskeleton. The heteroprotein can make the skeleton component appear more clearly.
Microfilaments, microtubules, intermediate fibers, and the like are structures having a small diameter. The largest single microtubule is only about 25 nm, which can only be seen under electron microscope. The main method for studying the cytoskeleton is to apply high-voltage electron microscopy or immunofluorescence microscopy. The cytoskeleton is observed by ordinary light microscope in this experiment. The fibers are bundled and the structure is dyed and has an exaggerated effect. Since the main component of the cells extracted by Triton X-100 is a cytoskeletal component, the appearance is equivalently clear.
Experimental reagent
M-buffer (preparation method is as follows):
Imidazole 2.90g
KCl 3.70g MgCl2 0.012g
EGTA 0.38g EDTA 0.042g
Mercaptoethanol 0.086ml Add water to 1000 ml
6 mM phosphate buffer
KH2PO4 0.74g
Na2HPO4?12H2O 2.4g
Add water to 1000 ml
1% Triton X-100: formulated in M-buffer
3% glutaraldehyde: 25% glutaraldehyde 12 ml 6 nm phosphate buffer 88 ml
Dyes: Coomassie Brilliant Blue R-250 0.2g Glacial Acetic Acid 7 ml
Methanol 46.5 ml distilled water 46.5 ml
experiment apparatus
Equipment: microscope, beaker, straw, tweezers, slides, coverslips
Experimental Materials
Material: onion bulb
Experimental procedure
1. Tear off the epidermal cells (about 1 cm2) in the onion bulbs and place them in a 6 mH pH 6.8 phosphate buffer to allow them to sink.
2. Aspirate the phosphate buffer and add 1% Triton X-100 for 20-30 minutes.
3. Aspirate Triton X-100 and wash three times with M-buffer for three minutes each time.
4.3% glutaraldehyde was fixed for 30-60 minutes.
5. Wash the pH 6.8 phosphate buffer three times for ten minutes each time.
6.0.2% Coomassie Brilliant Blue R-250 stained for 20-30 minutes.
7. Wash with distilled water 1-2 times, place the cells on a glass slide, add a cover glass, and observe under a common optical microscope, you can see a network composed of microfilaments and microtubules.
December 07, 2023
