Formaldehyde

Mouse bone marrow macrophages (BMM) from 8 wk old wild mice (Sino-British) were cultured in a T75 medium flask (Costar) (20 ml final volume) containing 30 ng/ml mCSF (R&D Industries, Minneapolis, MN, USA) in endotoxin-free RPMI-10% heat-inactivated fetal bovine serum (FBS) (Gibco). The cells were cultured for 3 days and non-adherent BMM precursors were collected by centrifugation at 1,500 g for 4 min at 4 °C. The cells were re-suspended in αMEM medium (Gibco) and cell counts were adjusted to 50 × 10⁵ BMM/ml. Then, they were seeded at 10⁵ cells/well/0.2 ml medium in 48 well plates and treated with RANKL (100 ng/ml)/mCSF (30 ng/ml) (R&D Industries) (control group), mouse oncostatin M (m-OSM)-RANKL-mCSF (positive control), collagen and polypeptides (1, 10 and 50 μg/ml)-RANKL-mCSF. The plates were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 7 days. Cells were fixed in 4% formaldehyde (Sinopharm) for 3 min followed by ethanol: acetone (1:1) for 1 min. After brief air dry, the cells were incubated with tartrate-resistant acid phosphatase (TRAP) stain (Sigma) for 8 min^{29 (link)}, and the number of double and triple plus nucleated TRAP-positive osteoclasts were counted under a light microscope (Olympus Inc.; Center Valley, PA, USA).

To observe the microstructure of each cell, samples in the glass-bottom dishes were fixed with freshly prepared 4% formaldehyde (Sinopharm Chemical Reagent Co. Ltd.) in PBS for 10 min at room temperature. After fixation, we washed samples with PBS for three times and added 0.1% Triton X-100 (Sigma Aldrich Co. Ltd., Shanghai) in PBS with 1% bovine serum albumin (MesGen, Shanghai Hongsheng biotechnology Co. Ltd) to each for 5 min. After this permeabilization, fluorescein-phalloidin solution (MesGen MF8203, Shanghai Hongsheng Biotechnology Co. Ltd) was applied for 20 min at room temperature inside a covered container during the incubation. The samples were subsequently washed with PBS for three times. We followed the protocol recommended in the product Hoechst 33342 (Sigma Aldrich Co. Ltd, Shanghai) for the cell nucleus staining. Confocal images were taken using a laser scanning confocal microscope (Olympus FV1200, Japan).

Mouse heart tissue was fixed in 4% formaldehyde (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) at room temperature for more than 12 h, then embedded in paraffin after complete dehydration using ethyl alcohol. Blocks were cut into 4-μm-thick slices, air-dried, dewaxed in xylene, and stained with hematoxylin and eosin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). Heart tissue images were captured using a fluorescence microscope (Olympus FluoView™ FV1000; Olympus Corp.) and light-field observation model.

A migration assay was performed in a Modified Boyden Chamber (Costar, #3422, Cambridge, MA) to examine cell migration. A total of 3×10⁴ cells were suspended in 200 μL serum-free DMEM and seeded onto polycarbonate filters for the migration assay; each lower chamber was filled with 600 μL of 10% FBS-DMEM. For the invasion assay, the top chamber membrane was coated with 40 μl of 0.125 mg/ml matrigel in serum-free DMEM and incubated at 37 °C for 2 hrs before use. To assess the ability of the GC cells to cross the polycarbonate membrane, 5×10⁴ cells in 200 μl of serum-free DMEM were placed into the upper compartment of the wells that were coated with the reconstituted Matrigel, and 600 μl 10% FBS-DMEM was placed into the lower compartment. After 24 hrs of incubation, cells that had migrated or invaded into the lower chamber were fixed for 10 min with 1 ml of 4% formaldehyde (Sinopharm, China) and stained with 0.5% crystal violet for 30 min. After removing the non-migrating cells, the migrated and invaded cells were photographed by an inverted light microscope (magnification, 200×, Nikon Corporation, Japan) at least 6 random fields for each well.

Ethanediamine (EDA, 99%), formaldehyde (30–40%), ethyl alcohol (99.7%), 3-aminophenol (99%), ethylenediamine tetraacetic acid disodium salt (EDTA·2Na, 99%), H₂SO₄ (95–98%), HCl (36–38%), NH₃·H₂O (25–28%), and BaCl₂·2H₂O (99.5%) were purchased from Sinopharm Chemical Reagent (Shanghai, China). NaOH (96%) and Na₂SO₄ (99%) were purchased from Shanghai Dahe Chemicals (Shanghai, China). NaH₂PO₄·2H₂O (99%), and Na₂HPO₄·12H₂O (99%) were purchased from Chinasun Specialty Product (Changshu, China). Tetraethoxysilane (TEOS, 99%), catalase (from bovine liver, 2000–5000 units/mg protein), 3,3′,5,5′-tetramethylbenzidine (TMB, 98%), and peroxidase from horseradish (HRP, ~200 units/mg) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All chemicals were used without further purification.

Alkali lignin (AL), product number L0082, was purchased from TCI Shanghai Co., Ltd. (Shanghai, China). The lignin was washed with aqueous 2 M hydrochloride acid and filtered in a sand core filter funnel, and then repeatedly washed with excess water until neutral. The filtered lignin was dried overnight in a vacuum oven at 40 °C and stored in a tightly sealed container. HBr (48 wt.%), HI (48 wt.%), phenol, formaldehyde (37 wt.%), ether, pyridine, acetic anhydride, N,N-dimethylformamide (DMF) and sodium hydroxide were AR grade reagents and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).