Alcian blue 8gx

To evaluate larvae skeletal anomalies and developmental status, 100 larvae/tank were stained with an acid-free double staining protocol for cartilage and bone adapted from Gavaia et al. [57 (link)] and Walker and Kimmel et al. [58 (link)]. Whole-mount acid-free double staining was performed using alcian blue 8GX (Sigma-Aldrich, Madrid, Spain) for cartilage and alizarin red S (AR-S) (Sigma-Aldrich) for mineralized tissues [58 (link)]. Samples were stained in 0.1% alcian blue 8GX solution (dry weight/volume) with 60 mM MgCl₂ in 70% ethanol for 3 h and rehydrated with a decreasing concentration of ethanol (96% to 25%) for 2 h. Then, the samples were stained overnight with 0.05% AR-S in 0.5% potassium hydroxide (KOH) (Sigma-Aldrich). Stained samples were cleared with 1% KOH and subsequently transferred through an increasing concentration of glycerol (25% to 100%). Samples were stored in 100% glycerol (Merk Millipore, Billerica, MA, USA) until observation.

Differentiated cell cultures were stained for the presence of mineral deposits using von Kossa and alizarin red S (Sigma) as described in previous studies (38 (link)). Briefly, for von Kossa staining, cells were rinsed three times with PBS, fixed in 10% neutral formalin buffer for 2 h, and then washed three times with distilled water. The fixed cells were stained with 2.5% silver nitrate solution for 30 min in the presence of light produced from a 100-watt incandescent light bulb and then washed three times with distilled water. The amount of Ca²⁺ in the matrix was measured using the Arsenazo III (Sigma) staining method described previously (39 (link)). To visualize acidic proteoglycans secreted by chondrocytes, we stained 10% formalin-fixed cells with 1% Alcian blue 8GX (Sigma; 1 g of Alcian blue 8GX dissolved in 100 ml of 0.1 n HCl) for 30 min and then washed twice with 0.1 n HCl. All of the fixed and stained cells were air-dried, and the images were captured using an Epson Perfection V750 flat-bed light scanner.

Postnatal day 2 (P2) mice were fixed in 95% ethanol for 24 hours with agitation. Skin was removed and organs were eviscerated. The mice were placed in Alcian Blue 8GX (Millipore‐Sigma) cartilage staining solution for 1 week and dehydrated in 95% ethanol for 1 week. Soft tissues were cleared with 1% potassium hydroxide (KOH) solution for 3 to 4 days and placed in Alizarin Red (Millipore‐Sigma) bone‐staining solution for several days until staining was complete. Soft tissues were further cleared in graded KOH solutions. Samples were stored in glycerol–formaldehyde solution before acquiring images.

Hyaluronic acid sodium salt (from Streptococcus equi bacterial glycosaminoglycan polysaccharide), 5-bromo-2'-deoxyuridine (BrdU), and Alcian blue 8 GX were purchased from MilliporeSigma (USA). Anti-BrdU, anti-aggrecan, and anti-Sox9 were purchased from GeneTex (Taiwan). Anti-phospho-Stat3 (Tyr705) was purchased from Cell Signaling Technology (USA). PE-conjugated anti-rat CD90 antibody was purchased from Abcam (USA). Atglistatin, SC-1, and cryptotanshinone (CPT) were purchased from Selleckchem (USA). Also, 29-mer and a control peptide (PEDF Glu97-Ser114) were modified to increase stability by acetylation at the NH₂ terminus and amidation at the COOH terminus and synthesized to the purity > 95%, determined using mass spectrometry by the vender (GenScript, USA).

Whole-mount skeletal stains were prepared using a modified published protocol (McLeod, 1980 (link); Rigueur and Lyons, 2014 ). Briefly, E15.5-E16.5 mouse embryos were harvested and the skin and internal organs were removed to facilitate tissue permeabilization. The embryos were then fixed first in 95% ethanol overnight at room temperature followed by 100% acetone overnight at room temperature. To stain the cartilage, the embryos were incubated overnight at room temperature in 0.03% (w/v) Alcian Blue 8GX (Millipore Sigma) prepared in a solution of 80% ethanol and 20% glacial acetic acid. After visually confirming that the embryos were completely blue, the embryos were destained in a series of ethanol washes (2-3 h in 100% ethanol, 75% ethanol, 50% ethanol and 25% ethanol). To stain the bone, the embryos were then incubated overnight at 4°C in 0.005% (w/v) Alizarin Red S (Millipore Sigma) prepared in 1% potassium hydroxide (KOH). The tissue was then cleared in 0.3% KOH for 1 to 3 days (changing the solution every day). Once the embryos cleared to the desired amount, the 0.3% KOH was replaced with glycerol. The embryos were transitioned through a series of glycerol solutions (20% for 1 day, 50% for 1 day and then 80% for 1 day). The skeletons were then kept in 80% glycerol for prolonged storage.

Cells were washed by PBS twice and then fixed with 4% formaldehyde for 2 min at room temperature. Formaldehyde was removed and cells were washed with ddH₂O. 1% Alcian blue 8GX (Sigma, 66011) was used to stain cells for 1h at room temperature. After staining, cells were washed with acetic acid twice and then quickly with ddH₂O. The plate was air dried and then digitally imaged (Epson Perfection V800 photo). For quantification, 1% SDS was added to the well and the plate was placed on a shaker overnight at room temperature. One hundred microliters of the eluate was transferred to a well of a 96-well plate (Corning, 3361) and the absorbance was measured at 605 nm (BioTek, Synergy hybrid H4 Reader).

Beads were removed from culture, washed in PBS, and fixed in 4% PFA solution containing sodium cacodylate barium chloride (0.05 M) buffer overnight at 4°C. After removing the fixative and washing, samples were gradually dehydrated through 30–100% ethanol series with a final xylene change, before embedding in wax. Sections of 8 μm were obtained with a microtome (Leica RM2125RT, Ireland) and affixed to microscope slides (Polylysine™, VWR, Ireland). Prior to staining, sections were dewaxed and rehydrated in 100% to 70% ethanol baths followed by distilled water. Cellular colonization and matrix deposition were observed using hematoxylin and eosin (H&E), sGAG deposition was evaluated using aldehyde fuchsin and 1% alcian blue 8GX in 0.1 M HCl, and collagen distribution was assessed using picrosirius red (all from Sigma-Aldrich, Ireland). Semiquantitative analysis of percentage (%) chondron in constructs was determined using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).