Protocol for Microfilaments and Cell Morphology

• The longitudinal and transverse fiber network in eukaryotic cytoplasm is called the cytoskeleton, which plays an important role in maintaining cell shape and movement. Depending on the composition and assembly structure, the cytoskeleton can be divided into microtubules (MT), microfilaments (MF), and intermediate fibers (IF). Microfilaments are prevalent in a wide range of cells and have a role in cell shape and movement.
• When the cells are treated with TritonX-100 solution, it could dissolve many proteins in the plasma membrane structure and intracellular, while the proteins in the cytoskeleton are not destroyed. After fixation and staining with Caulmers Brilliant Blue (a non-specific protein dye), the cytoplasmic background is weakly stained, and the microtubules and other protein structures cannot be differentiated under the light microscope. Stress fibers composed of microfilaments are mainly observed under the light microscope.
• Stress fibers are composed of microfilaments arranged in parallel, and they are specially developed in adherent cells cultured in vitro, with a long and straight morphology, often parallel to the long axis of the cell and running through the entire length of the cell.

• When fibroblasts are subjected to passaging culture, sterilized coverslips are coated with polylysine, placed in Petri dishes, and grown for 24-48 h in a 37°C, 5% CO₂ temperature chamber.
• Remove the coverslip with cells, put it into a small culture (front side up), and wash it with PBS 3 times (gently add 3-4 drops from the corner of the coverslip) to wash off the surface of the culture solution.
• Discard the PBS, add 4-5 drops of 1% Triton X-100 solution (just covering the surface of the coverslip), and process for 15 min at room temperature.
• The Triton X-100 is aspirated and immediately washed gently 3 times with an M-buffer.
• The fixation is dried slightly and fixed in 3% Chengdialdehyde solution for 10 min, then washed 3 times with PBS solution to wash off the fixative.
• Stain with 3-5 drops of 0.2% Caumas Brilliant Blue Staining Solution for 10 min, then carefully rinse with tap water.
• A small amount of water is retained to seal the slide, and absorbent paper is used to absorb the excess water. The clinical slide is observed under a light microscope.

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• Wash the slides gently so as not to wash the cells off the mount.
• Recovery time should be sufficient or the cells will not return to their condition before treatment.
• Pay attention to the front and back of the cell cover slip.
• The back side of the cover sheet should be rinsed after staining to avoid damaging the cells.

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