This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare 1-10 mg/mL Alcian Blue 8GX staining solution: add 0.6 mL glacial acetic acid to ddH2O to make the final volume 20 mL. Then add 20-200 mg of solid Alcian Blue 8GX, fully dissolve and then use a 0.22μm filter to filter and sterilize.
Note: The prepared dye solution should be stored at 4℃ away from light and is recommended to be used within one month.
2. Cell suspension staining (taking 6-well plate as an example)
(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.
(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.
(3) Add 1 mL of dye working solution to resuspend the cells, and incubate at room temperature in the dark for 30-60 minutes. The optimal culture time for different cells is different.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS to wash 2-3 times, 5 minutes each time.
(5) Resuspend the cells in serum-free cell culture medium or PBS and observe through a microscope.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 30-60 minutes.
(4) Aspirate away the dye working solution and use culture solution to wash the coverslip 2 to 3 times for 5 minutes each time.
4. Microscopic examination: Use a light microscope to observe the stained cells.
Note: For your safety and health, please wear a lab coat and disposable gloves.
References:
[1]. Hiroki Ueharu,et. Isolation and Culture of Cranial Neural Crest Cells from the First Branchial Arch of Mice. 2022 Apr 5;12(7):e4371. doi: 10.21769/BioProtoc.4371.
[2]. J Tas. The Alcian blue and combined Alcian blue--Safranin O staining of glycosaminoglycans studied in a model system and in mast cells. 1977 Mar;9(2):205-30. doi: 10.1007/BF01003632.
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