Sodium azide

Chemicals used for the staining of tissue sections were as follows: hydrogen peroxide, hydrochloric acid (HCl), methanol, Permount, polyvinylpyrrolidone (PVP) (Cat.#BP431-100), SafeClear Xylene Substitutes, Triton X-100, and potassium ferrocyanide trihydrate (Cat.#S25489) were purchased from Fisher Scientific (Hanover Park, IL). Sodium azide, sodium borohydride, proteinase K, phosphate-buffered saline (PBS), Trizma base, and 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Cat.#D5905) were purchased from Sigma (St. Louis, MO). Chemicals used for test tube studies were as follows: Ferric iron solution (iron Standard for AAS, 1,000 mg/L Fe in nitric acid), Sodium azide, and DAB were purchased from Sigma-Aldrich (Oakville, Ontario, Canada), whereas K₄Fe(CN)₆ (EM Science), TRIS (EM Science), HCl (BDH), and H₂O₂ (BDH) were purchased from VWR (Mississauga, Ontario, Canada). Proper attention to safety is required when handling these chemicals.

Histone H1, 2,4-dinitrophenyl hydrazine (DNPH), 1-anilinonaphthalene-8-sulfonic acid (ANS), sodium dodecyl sulfate (SDS), methylglyoxal, aminoguanidine hydrochloride, diethylene triamine penta-acetic acid (DTPA), sodium azide, ethidium bromide, protein A-agarose (2.5ml pre-packed column), agarose, sodium azide, Tween-20, dialysis tubings, anti-human and anti-rabbit IgG, alkaline phosphatase conjugate, para-nitrophenyl phosphate, Freund’s complete and incomplete adjuvants were purchased from Sigma Chemical Company, St. Louis, MO, USA. Acrylamide, bisAcrylamide, ammonium persulfate (APS) and N,N,N′,N′- tetramethylethylenediamine (TEMED) were from Bio-Rad Laboratories, U.S.A. Sodium hydroxide, Ethylenediaminetetraacetic acid (disodium salt), methanol, glacial acetic acid, iso-propanol, sodium chloride, sodium carbonate, sodium nitrite, silver nitrate, xylene, sodium hydroxide, formaldehyde, sodium bicarbonate, ethanol, ammonium sulphate and ammonium persulphate were obtained from Qualigens, India. Polystyrene microtitre flat bottom ELISA plates and modules were purchased from NUNC, Denmark. All other chemicals/reagents were of the highest analytical grade available.

Frozen breast tumors were decellularized as described [18 (link),22 (link)]. In brief, tumors were decellularized in two rounds of lysis buffer containing 5 mM EDTA, 0.1% sodium dodecyl sulfate, 0.4 mM phenylmethylsulfonyl fluoride (PMSF) and 0.02% sodium azide (all Sigma-Aldrich, St. Louis, MO, USA) in water for 6 h, followed by a rinse step with water supplemented with sodium azide, EDTA and PMSF at the same concentrations for 15 min. During the following 72 h, PDSs were washed with H₂O and, thereafter, for 24 h with phosphate buffered saline solution (PBS; Medicago) to remove cellular debris. Decellularization was performed at 37 °C and agitation at 175 rpm (Incu-ShakerTM 10 L, Benchmark). Patient-derived scaffolds were then placed in a storage solution containing sodium azide, EDTA and PBS at 4 °C to preserve the tissue until usage. Patient-derived scaffolds were mechanically made into even sections using biopsy punch needles of Ø 6 mm. Samples were then snap-frozen in liquid nitrogen and sectioned to 150 µm thin slices using CM3050 S (Leica) cryotome. The PDS slices were then sterilised in PBS supplemented with 0.1% peracetic acid (Sigma-Aldrich) for 1 h at room temperature, followed by 24 h wash with PBS containing 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA), at 37 °C and 175 rpm.

For direct detection of cell surface proteins, cells were incubated with fluorescently labelled antibodies for 30 mins at 4°C or 15 mins at room temperature. Labelled cells were washed twice with FACS wash (PBS, 1% w/v BSA (Europa Bioproducts), 0.01% v/v sodium azide (Sigma-Aldrich) and centrifuged at 300 × g for 5 mins. For indirect detection of hIgG bound to the cell surface, target cells were opsonised with mAb for 30 mins at 4°C or 15 mins at room temperature, washed twice with FACS wash, 0.01% sodium azide (Sigma-Aldrich) at 300 × g for 5 mins, and labelled with mouse anti-human IgG-APC (clone M1310G05) for 30 mins at 4°C or 15 mins at room temperature. Labelled cells were washed twice with FACS wash at 300 × g for 5 min. Samples were analysed using a FACS Calibre or Canto (Becton Dickinson) and data analysis was performed using FlowJo (Becton Dickson).

For analysis of IRF5 translocation, DCs or macrophages were stimulated as indicated in Maxisorp plates. After 2 h stimulation, cells were washed with PBS, fixed with 3.7% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed in PBS and stored in PBS containing 0.5% bovine serum albumin (BSA; PAA) and 0.1% sodium azide (Merck) at 4°C. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked for 30 min in PBS containing 0.5% BSA and 0.1% sodium azide. Cells were then stained with a rabbit-anti-human-IRF5 antibody (1:400) (Cell Signaling) or rabbit-anti-human NF-kB p65 antibody (1:100) (Cell Signaling) for 45 min at room temperature, washed with PBS and stained with a Cy3-labeled goat-anti-rabbit-IgG antibody (1:50) (Jackson ImmunoResearch). Cells were again washed with PBS and nuclei were stained using 1 μg/mL Hoechst (Immunochemistry Technologies) for 1 min at room temperature. Cells were imaged using a DM IRB inverted fluorescence microscope (Leica), combined with a DFC 300FX digital color camera (Leica).

Mice were euthanized with isoflurane (5%, Panion & BF Biotech), and subsequently perfused transcardially with PBS and paraformaldehyde (4% w/v, Electron Microscopy Sciences) for brain collection. The brain samples were dissected and post-fixed in 4% w/v paraformaldehyde for 3 days at 4 °C. After 3 days, the brain samples were washed with PBS. The samples were then immersed in PBS with 0.02% sodium azide (Sigma-Aldrich) at 4 °C until sectioning. For the purpose of the c-Fos labeling for neural activation, mice were sacrificed and sampled 80 min after the behavior test. The brain samples were embedded in the 4%UltraPure low melting point agarose (Invitrogen) in PBS on the ice. Then the solidified embedding samples were sectioned coronally for 50 μm by using the vibratome (Leica). The free-floating brain slices were kept in PBS with 0.02% sodium azide (Sigma-Aldrich) at 4 °C until immunohistochemistry staining.

The ability of the plasmids harboring the mcr genes and the bla_VIM-1 gene to conjugate was tested through conjugation experiments. The conjugation was performed in Mueller Hilton (MH) broth (OXOID, Hampshire, UK) using E. coli A15^rAzi as the recipient.
Transconjugants for ENCL_3849 were selected on MH agar (OXOID, Hampshire, UK) plates supplemented with sodium azide (150 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and ampicillin (1000 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). For ENCB_IB2020, transconjugants were selected on MH agar plates supplemented with sodium azide (150 mg/L), meropenem (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and colistin (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). The presence of bla_VIM-1, and mcr-like genes in the transconjugants was confirmed through PCR. MICs for transconjugants were performed using the broth-microdilution method. Isolates that failed to transfer the mcr genes of interest through conjugation were subjected to transformation; plasmids were extracted using Qiagen Maxi kit (Qiagen, Hilden, Germany) and the competent E. coli DH5α cells were used as the recipient. Transformants were selected on MH agar (OXOID, Hampshire, UK) with 2 mg/L colistin. Transformants were confirmed to be MCR producers through PCR.