Alcian Blue 8GX

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Alcian Blue 8GX staining of cell micromass cultures^[1]
1.1 Prepare preprocessing solution A: add 0.5 mL of ammonium hydroxide solution to 500 mL of 70% ethanol.
1.2 Prepare preprocessing solution B: add 25 mL of glacial acetic acid to 70% ethanol to a final volume of 500 mL.
1.3 Discard the culture medium from the micromass culture and fix with 1 mL of Bouin's fixative at room temperature for 15 minutes.
1.4 Wash with preprocessing solution A and B.
1.5 Stain with 2 mL of 1 mg/mL Alcian Blue 8GX at room temperature for 1 hour.
1.6 Wash off the dye with deionized water, repeat four times. and soak in 2 mL of deionized water.
1.7 Take a photograph.
1.8 Wash off the dye again with deionized water.
1.9 Extract the dye by incubating the culture with 1 mL of 6 M guanidine hydrochloride at room temperature overnight.
1.10 Measure the optical density at OD595 using a spectrophotometer.
2. Cell Culture Precautions^[1]
2.1 Avoiding Cell Death: To prevent cell death, embryos/cells should always be kept on ice before plating.
2.2 Handling When Cell Viability is Below 70-80%: If cell viability drops below 70-80%, consider using a dead cell removal kit (e.g., Miltenyi Dead Cell Removal Kit, Miltenyi Biotech, Cat. No: 130-090-101) to eliminate dead cells.
2.3 Cell Plating in 35 mm Culture Dishes: When plating cells in 35 mm culture dishes, maintain a high-density cell area. After 1.5 hours of culture, the cells should adhere. Carefully add high DMEM culture medium containing fibronectin.
2.4 Use and Passaging of Isolated Cells: Isolated cells can be used for micro-clump culture on the same day they are isolated. However, these cells can only be passaged a maximum of three times, and the total culture time from isolation should not exceed 10 days.
3. Alcian Blue 8GX staining of glycosaminoglycans^[2]
3.1 Mast cells are fixed with methanol-40% formaldehyde-acetic acid (85:10:5 volume ratio) for 30 min, and the cells are stained after washing.
3.2 Stain with 0.5% (w/v) Alcian Blue 8GX dissolved in 3% (v/v) acetic acid, pH 2.4, for 0.5 or 2 hours.
3.3 The same solution saturated with urea as in 3.1 is returned to pH 2.4 with HCl and stained for 0.5 h.
3.4 After staining with the same solution as in 3.1 for 0.5 h, the slides are stained with 0.3% (w/v) hematoxylin O (pH 0.9) in 0.125 N HCl for 5 min.
3.5 After staining, rinse with distilled water, let it air dry, and then mount with Entellan.
3.6 To detect the presence of histamine, air dry the unfixed or fixed smears of rat peritoneal mast cells, and stain them with 1% (w/v) ortho-phthalaldehyde (OPT) dissolved in xylene (staining time 2 minutes). The staining process is carried out in a closed chamber at room temperature with 100% relative humidity. After staining, the slides are drained and immediately mounted with tetrahydrofuran.
3.7 The yellow fluorescence intensity of the histamine-OPT complex in mast cell granules is examined using a Leitz Orthoplan microscope equipped with a Leitz Ploemopak 2 (for incident fluorescence) and a 75 W high-pressure xenon lamp. The filter system used consisted of a TK 510/K 515 dichroic, a K 510 blocking filter and a 2 x KP 490 (= KP 500) excitation filter.
4. Alcian Blue 8GX staining of fetal cartilage^[3]
4.1 Rat fetuses are obtained from mated female Spraque-Dawley rats. On gestational day 21 (sperm/sperm day 5, gestational day 0), rats are euthanized by carbon dioxide asphyxiation. Fetuses are obtained by standard cesarean section and subsequently euthanized by oral administration of sodium pentobarbital. New Zealand white and Dutch rabbits are 5-6 months old at the time of mating, and pregnant rabbits are sacrificed on gestational day 28 and fetuses are obtained by intravenous injection of sodium pentobarbital. Each fetus is euthanized by oral administration of sodium pentobarbital.
4.2 Fetuses are eviscerated, skinned, and fixed in 70% ethanol for 3-7 days.
4.3 Alcian Blue 8GX is added to a mixture of 8 parts 95% ethanol and 2 parts glacial acetic acid to prepare solutions of 75 and 100 mg/L.
4.4 Rat fetuses are stained in Alcian Blue 8GX (100 mg/L) for 24 h and rabbit fetuses are stained in Alcian Blue 8GX (75 mg/L) for 24 h, followed by rinsing in 95% ethanol for 6 h.
4.5 Rat and rabbit fetuses are immersed in 1% and 1.25% potassium hydroxide, respectively. Rat fetuses are stained with 12 mg/L Alizarin Red S (HY-120601) for 48 h and rabbit fetuses are stained with 6 mg/L Alizarin Red S (HY-120601) for 24 h.
4.6 Transferred to a solution of 70% ethanol and glycerol (1:1) for clearing, hardening, and examination.
5. Alcian Blue 8GX for protein determination^[4]
5.1 Dissolve the target protein in water or 0.1 M NaCl solution to a working concentration of 25 μg/ml.
5.2 Use Britton-Robinson buffer (pH 7.24, containing 0.04 M H3PO4, 0.04 M HAc and 0.04 M H3BO3) to control the acidity and use 0.5 M NaCl solution to adjust the ionic strength of the aqueous solution.
5.3 Add the appropriate protein working solution, 1.0 mL of Britton-Robinson buffer. Except for IgG, which was diluted with 0.1 M NaCl, all other mixtures were diluted to 10 mL with double distilled water.
5.4 The mixture is used for absorption or RLS measurement. The RLS spectrum is scanned by adjusting the excitation and emission monochromators (?λ = 0 nm) of the RF-540 fluorescence spectrometer. The RLS intensity is measured at 398.0 nm, proteins, including bovine serum albumin (BSA), human serum albumin (HAS), pepsin (Pep) and cellulase (Cel), can be determined with the limit of detection below 30 ng/mL. 5.5 RLS spectra of the interaction between ABGX and BSA. Concentration 1.0 × 10^-5 M.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.