Ethylene diamine tetraacetic acid (edta)

Ultrapure water with conductivity of 5–15 M Ω cm⁻¹ (Millipore, Bedford, MA, USA) was used for making up volume standards and reagents.
Standard solutions of 1000 mg L⁻¹ Sb(III) and Sb(V) were prepared from C₈H₄K₂O₁₂Sb₂ xH₂O (Fluka, Neu-Ulm, Germany) and KSb(OH)₆ (Riedel de-Haën, Seelze, Germany), respectively. A certified standard solution of 1000 ± 4 mg L⁻¹ antimony, prepared from 99.999% “purum” metallic Sb, dissolved and stabilized in high-purity acids (5% nitric acid (HNO₃) and 0.1% hydrofluoric acid (HF)), was used to standardize both standards. All standard solutions were kept refrigerated in high-density polyethylene bottles. The media of the daily working standard solutions were diluted acid and mobile phase for total and speciation Sb analysis, respectively.
A 10 mM EDTA (Panreac, Barcelona, Spain) solution adjusted to pH 4 with diluted ammonia (Panreac) was filtered daily through a 0.45 μm filter (Millipore type HA) for use as the mobile phase.
LC/MS grade methanol and water (Fluka, Madrid, Spain) were used to prepare the solutions for mass spectrometry analysis.

Palmitoyloleoylphosphatidylcholine (POPC) was purchased from Avanti Polar Lipids (USA); Phospholipase A2 from bee venom was obtained from Sigma-Aldrich. Enzyme stock solution (1.0 mM) was prepared in 0.1 M Tris-HCl buffer containing 0.1 M NaCl, pH 8.5. Enzyme concentration was controlled by the absorption at 280 nm (Nano Drop OneC, Thermo Fisher). 2-Amino-2-(hydroxymethyl)propane-1,3-diol (Tris base, Fluka), HCl, NaOH, ethylenediaminetetraacetic acid (EDTA, Panreac), and CaCl₂ were of reagent grade.

CTAB (CAS: 57-09-0), was purchased from Acros Organics (Portugal), while cinnamaldehyde (CAS: 14371-10-9) and bovine serum albumin (BSA) were purchased from Sigma Aldrich (Portugal). EDTA was acquired from Panreac. Lecithin, polysorbate 80, thiosulphate, saponin, isopropanol, and DMSO were obtained from VWR Chemicals. L-histidine was purchased from Merck. All reagents were of analytical grade. Solutions of CTAB, BSA, and EDTA were prepared in sterile deionized water and cinnamaldehyde in DMSO or isopropanol. The biocide and phytochemical neutralization step was performed using the universal neutralizer (Lecithin 3 g L⁻¹, polysorbate 80 30 g L⁻¹, thiosulphate 5 g L⁻¹, L-histidine 1 g L⁻¹, and saponin 30 g L⁻¹ in 1% phosphate buffer 0.25 M, pH 7.2) for 10 min, which was shown to be efficient in inactivating the biocidal activity and to be nontoxic to the test bacteria (data not shown).

All solutions were prepared using ultrapure water of resistance 18 MΩ cm^-1 (produced using a Milli-Q purification system, Millipore Corp., Bedford, MA). Stock standard solutions of the elements (1000 mg L^-1) were of ultrapure grade: ICP Multi element standard solution IV certiPUR for B, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Sr and Zn; and ICP standard certiPUR for Hg and Se were both purchased from Merck (Poole, U.K.). As, Mo, Sb and U standards were obtained from Panreac (Barcelona, Spain).
In the SAD preparation method, the samples were digestion in nitric acid (69%, Hiperpur-Panreac, Barcelona, Spain) and hydrogen peroxide (33% w/v, Panreac, Barcelona, Spain). In the AKD preparation method, the samples were digested in a mixture of ammonia (NH₄OH, 25%, Merck, Darmstadt, Germany), Triton X100 (Panreac, Barcelona, Spain), anhydrous 1-butanol (Panreac, Barcelona, Spain) and EDTA (Panreac, Barcelona, Spain). The certified reference material (NIST SRM-1598a inorganic constituents in animal serum) used to validate ICP-MS measurements was obtained from the National Institute for Standards and Technology, NIST (Gaithersburg, MD, USA).
Polypropylene tubes used for preparation of samples and standards were soaked in 10% Hiperpur HNO₃ for at least 24 h and rinsed with deionised water and dried before use. The sample tubes were tested and found to be free of trace elements.

Quant-iT PicoGreen dsDNA assay kit (Invitrogen, USA) was used to quantify residual DNA in the specimen. Similar to the previously published protocol^{22 (link)}, the specimen was lyophilized at − 40 °C for 24 h and its dry weight was measured. The dried specimen was then incubated in a papain solution, which contained 20 U/mL papain (Worthinton, Lakewood, NJ), 1.1 mM EDTA (Panreac, Spain), 5.5 mM cysteine-HCl (Panreac, Spain) and 0.067 mM 2-mercaptoethanol (Alfa Aesar, England) overnight at 60 °C until the HUA was completely digested. The solution was diluted with 0.2 M Tris–EDTA buffer and then incubated with the working solution of the kit in a 96-well plate. The fluorescence of the sample was measured using a fluorometer (excitation: 485 nm, emission: 538 nm; Fluoroskan Ascent, Thermo Fisher Scientific, Waltham, MA) and compared with that of a λ dsDNA standard (0–10 ng/mL) to determine the weight of residual DNA in the HUA. Finally, the weight of the residual DNA was normalized by the dry weight of the specimen.

When glial cell cultures were confluent, astrocytes were removed by mild trypsinization (0.125 g/L trypsin from porcine pancreas (T7409, Sigma-Aldrich, St. Louis, MO, USA) and 0.05 g/L EDTA (131669, Panreac, Darmstadt, Germany) in MEM). The adherent microglial cells were maintained in culture, in M10C-G medium, for a further five to seven days until use. Immunocytochemistry analysis showed that about 97.9% of the cells in the culture were positive for ionized calcium-binding adaptor molecule 1 (Iba-1) (Figure S3).

The samples were fixed in 10% formalin, decalcified in EDTA (PanReac AppliChem, Darmstadt, Germany), frozen in O.C.T. (Sakura Finetek Japan Co.,Ltd., Tokyo, Japan), and then sectioned at 30 µm. The sections were stained by Hematoxylin and Eosin, Safranin O, and Fast Green (all Merck KGaA, Darmstadt, Germany). To assess the collagen fibers organization in newly formed tissues we used the staining with 0.1% solution of Direct Red 80 (365548-5G Merck KGaA, Darmstadt, Germany) in 1.3% picric acid aqueous solution. The sections were analyzed with the polarizing microscope [31 ]. Collagen I fibers form tightly packed bundles with strong birefringence and appear orange in polarized light. Collagen III fibers form thin bundles with weak birefringence and appear green in polarized light [31 ].