Marking method of active product spherulous cell in anixella sp.
A technology for storing cells and labeling methods, which is applied in the field of active product storage cells and chemical staining and labeling in small axis sponges, to achieve the effect of facilitating large-scale counting and statistics, accurate and reliable results, and saving operating costs and time.
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Embodiment 1
[0023] Active product storage cells (Sp cells) were revealed by DAB staining of spongy tissue after dispersal
[0024] Take fresh small shaft sponge tissue, soak it in CMFASW containing 10mM EDTA for 10 minutes, discard the soaking solution, add CMFASW containing 4% formaldehyde, gently squeeze the tissue block after 1.5 hours, centrifuge the obtained cell solution at 29g, discard Add CMFASW to the supernatant to become a cell suspension, take 20 μl smear, immerse in distilled water after drying, take it out, add DAB staining solution (DAB 10g / L, H 2 o 2 0.1%, Tris-Base 9.68g / L, pH7.4), dyed for 5min, washed off the dye solution with tap water, and observed under a light microscope, the cells containing dark brown stained granules were Sp cells ( figure 1 ).
Embodiment 2
[0026] DAB staining of different cellular components of the minor axis sponge
[0027] Different components obtained by cell separation: enriched Sp cells ( figure 2 -a), 29g centrifuged large cells (containing individual Sp cells, figure 2 -c) and small cells centrifuged at 110 g (without Sp cells, figure 2 -e), add CMFASW containing 2.5% glutaraldehyde, fix for 0.5 hours, centrifuge to collect cells, smear the cell suspension, soak in distilled water after drying to remove salt, add DAB staining solution (DAB 2.5 g / L, hydrogen peroxide 1%, Tris-Base 3.63g / L, pH7.2), stained for 8 minutes, washed off the staining solution, and observed with an optical microscope at a magnification of more than 400 times, the enriched Sp cells contained dark brown staining Dyed particles ( figure 2 -b), 500rpm large cells contain individual granular stained Sp cells ( figure 2 -d), 1000rpm small cells do not contain Sp cells (no staining, figure 2 -f); The protein extracts of these ...
Embodiment 3
[0029] DAB Staining of Cells During Culture of Minor Spongy Cell Mass
[0030] Take the small axis sponge cell mass cultured for 5 days, disperse it in CMFASW containing 30mM EDTA, add formaldehyde to a final concentration of 2%, fix the cells for 1 hour, centrifuge the cells, discard the supernatant, add CMFASW to become a cell suspension, and take 20μl smear , dry and immerse in distilled water, take it out and add DAB staining solution (DAB 5g / L, H 2 o 20.4%, Tris-Base 6.05g / L, pH7.2), stained for 5min, washed off the dye solution with tap water, and observed under a light microscope, the original cells that partially phagocytized the Sp cells were diffusely stained as a whole, which did not conform to the pattern of Sp cells. characteristics, easy to distinguish from Sp cells, and cells containing dark brown stained granules are Sp cells ( Figure 4 ). This example shows that the method for identifying Sp cells by DAB granular staining is not only applicable to the cell...
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