The vesicular lineage live staining assay in cells takes advantage of the property that neutral red (neutrol red) is a specific live stain for the vesicular lineage, and stains only the vesicular lineage red while the cell is in the living state, leaving the cytoplasm and nucleus unstained.

Principle

The basic principle of the vesicular lineage staining experiment in cells is that the vesicles wrapped by a single membrane in animal cells belong to the vesicular lineage, including Golgi complex, lysosomes, endoplasmic reticulum, transport vesicles, phagocytic vesicles and so on. Chondrocytes contain more rough endoplasmic reticulum and developed Golgi complex, can synthesize and secrete cartilage mucin and collagen fibers, etc., so the vesicle system is developed. Neutral red (neutrol red) is the vesicular system of specialized living stain, in the cell in the living state, only the vesicular system will be stained red, the cytoplasm and the nucleus is not stained.

Operation method

Live staining experiments of the vesicular lineage in cells

Principle

The basic principle of the vesicular lineage staining experiment in cells is that the vesicles wrapped by a single membrane in animal cells belong to the vesicular lineage, including Golgi complex, lysosomes, endoplasmic reticulum, transport vesicles, phagocytic vesicles and so on. Chondrocytes contain more rough endoplasmic reticulum and developed Golgi complex, can synthesize and secrete cartilage mucin and collagen fibers, etc., so the vesicle system is developed. Neutral red (neutrol red) is the vesicular system of specialized living stain, in the cell in the living state, only the vesicular system will be stained red, the cytoplasm and the nucleus is not stained.

Materials and Instruments

Equipment:
Dissecting equipment, wax disk, slides, coverslips, pipettes, absorbent filter paper, ordinary light microscope, toadstools.
Reagents:
① 1/3000 neutral red; ② 0.65% Ringer liquid (for amphibians)
② 0.65% Ringer's liquid (for amphibians) (NaCl, 0.65 g; KCl, 0.042 g; CaCl2, 0.025 g; distilled water, 100 ml).

Move

The basic procedure for in vivo staining of the vesicular lineage in cells can be divided into the following steps:
A Take a toad, execute it by smashing the spinal cord, fix it on a wax plate with the ventral surface facing upwards, cut open the abdominal cavity, and take the thinnest part of the cartilage of the sternocleidomastoid bone and put it on a slide.
B Apply 1/3000 drops of Neutral Red, and stain the slide for 15 min.
C Absorb the solution with a piece of filter paper.
D Apply 0.65% of Ringer's liquid drop by drop and put on a cover sheet, and suck excess liquid from the side of the cover sheet with a filter paper. Pipet the excess liquid from the side of the cover slip with filter paper.
E Observe under the microscope.

Caveat

1 In order to facilitate observation, when taking the sternal raphe, try to take the thinner part.2 As this experiment is a live staining, care should be taken to keep the specimen in a live state during the whole process of the experiment, especially when taking the material, it should be done accurately and quickly.

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