Copper sulphate

M. psychrotolerans was obtained from the German collection of microorganisms and cell cultures (DSMZ). Cells were grown in low-salt LB medium (5 g L⁻¹ yeast extract, 10 g L⁻¹ tryptone). For aerobic nanoparticle synthesis, cells were incubated at 20 °C in a shaking incubator (200 rpm) for 24 h. For anaerobic nanoparticle synthesis, low-salt LB medium was degassed and then M. psychrotolerans was incubated at 30 °C in an anaerobic cabinet under 10% CO2, 10% Hydrogen, Nitrogen Anaerobic Mixture at 200 rpm for 24 h. To produce copper nanoparticles copper sulphate (Sigma-Aldrich) was added to the growth medium to a final concentration of 5 mM. After this the cultures were incubated for a further 24 h, aerobically or anaerobically as required. After a total incubation of 48 h the cultures were centrifuged at 4000 rpm (3200g) for 10 m to pellet the cells. The supernatant was removed and filtered through a 0.22 μm filter to take out any remaining cells, with copper nanoparticles present in the filtered supernatant.

BLAST search on the NCBI website confirmed that the epitopes recognized by the antibodies used in this study (Table 1) are present with high homology in human and in degus. Antigen retrieval was conducted according to the antibody supplier’s instructions.
The primary antibodies (Table 2) were diluted in 3% NGS or 3% NDS in PBS and incubated overnight at room temperature. The negative control for the immunohistochemical procedure included omission of the primary antiserum, which was replaced by the antibody diluent followed by normal processing. The appropriate secondary antibodies were then applied for 3 hours at room temperature in the dark. The secondary antibodies used were goat anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 594 or 488 diluted to 1:500 using 3% NGS solution. Negative controls resulted in no labeling of the retina. The nuclear marker, 4',6-diamidino-2-phenylindole (DAPI, D9542, Sigma Aldrich), was also included in the secondary antibody solution at 0.125μg/ml. Quenching with copper sulphate [20% (w/v) Sigma] in water for an hour was a step in all immunohistochemical procedures before mounting the retina in the anti-fade medium, Citifluor.

CCl₄, reduced glutathione (GSH), glutathione reductase, γ-glutamyl p-nitroanilide, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, potassium iodide, sodium tartrate, copper sulphate, bromo cresol green, hydrogen peroxide solution, phenazine methosulphate, glycylglycine, magnesium chloride, guaiacol, sulfosalicylic acid, sodium azide, reduced nicotinamide adenine dinucleotide (NADH), sodium hydroxide, and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. USA.

The activity of secreted LsAA9A from K. phaffii was determined using a azurine cross-linked hydroxyethylcellulose (AZCL-HEC) chromagenic assay that enables rapid detection of enzymatic activity on polysaccharides^{51 (link)}. 1 mg/mL AZCL-HEC substrate (Megazyme, County Wicklow, Bray, Ireland) was mixed with with 1 mM ascorbic acid (Sigma-Aldrich, Saint Louis, MO, USA), 100 μM copper sulphate (Sigma-Aldrich), and the volume was adjusted with 100 mM sodium acetate (pH 5) (Sigma-Aldrich). 100 µL of the secreted LsAA9A sample was mixed with 400 μL of the AZCL-HEC substrate reaction and incubated at 50 °C with shaking at 1500 rpm for 1 h. The samples were centrifuged to remove the AZCL-HEC substrate, and the absorbance was measured at 590 nm.

Copper sulphate (CuSO₄, > 98%), lithium sulphate (Li₂SO₄, > 98%), 1,2-dichloroethane (DCE, ≥ 99.0%), 1-bromooctane (99%), trioctylphosphine (97%), and 2,2′:5′,2′′-terthiophene (TT, 99%) were acquired from Sigma-Aldrich. All reagents were used without additional purification. Ultrapure water from a MilliQ filtration system (> 18.2 MΩ cm) was used throughout to generate aqueous solutions. The tetraoctylphosphonium tetrakis(pentafluorophenyl)borate (P₈₈₈₈TB) ionic liquid used as an oil phase supporting electrolyte was prepared as detailed previously^{41 (link)}.