Glutaraldehyde

Phosphate-buffered saline (PBS) buffer compounds (NaCl, KCl, Na₂HPO₄, and KH₂PO₄), glucose, glutaraldehyde, paraformaldehyde, glycerol, N-ethylmaleimide, were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Ammonium phosphate monobasic, dithiothreitol (DTT) iodoacetamide, and trifluoroacetic acid (TFA) were supplied by Sigma Aldrich (St. Louis, MO, USA), and α-cyano-4-hydroxycinnamic acid (HCCA) was supplied by Bruker Daltonics (Bremen, Germany). Trypsin was supplied by Promega (Madison, WI, USA) Acetonitrile (ACN), 2-propanol, ethanol, and acetone were supplied by J.T. Baker (Center Valley, PA, USA). The reagents were of analytical grade or better. The water used in the study was of Milli-Q quality. Melittin, tertiapin and apamin were supplied by Sigma Aldrich (St. Louis, MO, USA).

Biofilms were formed as specified above in a glass coverslip. After the treatment with the test substance on a glass coverslip, the biofilm was fixed overnight with 3% (v/v) glutaraldehyde (VWR, Avantor, USA) solution in PBS at 4 ℃. Post-fixation of the biofilm was done by a 2% (v/v) osmium tetroxide solution (Sigma-Aldrich, Germany) for 1 h at room temperature. After each fixation, bacterial cells were washed twice with PBS and dehydrated with ethanol (VWR, Avantor, USA) 25%, 50%, 75%, and 100% for 15 min each. In addition, the samples were dried overnight using a freeze-drying process (BioGene lyophilizer freeze dryer, BioGene Biotechnologies, Ghaziabad, India). Each sample was mounted on double-sided tape glued to an aluminum supporter, and the samples were gold-covered using an Ion Coater (Quorum model Q 150R ES, Quorum Technologies, United Kingdom). Finally, the preparations were observed using a VP SEM Hitachi S-3400N (Hitachi Instruments, Inc., San Jose, CA, USA) scanning electron microscope (SEM).

The test organism Leishmania donovani (WHO strain DD8) was a gift from Dr. Neelo Singh of the Leishmania Research Society, India. Quinine sulphate standard was a gift from the Centers for Disease Control and Prevention, Atlanta, GA, USA. Bovine serum albumin (BSA) powder, culture media (M199), Alamar blue, glutaraldehyde, sodium bisulfite, and all other reagents used for experiments were purchased from VWR International (Radnor, PA, USA).

Whole blood clot formation and firmness were analysed using a ROTEM Delta (Werfen Limited, Warrington, UK). All investigations were performed through activation of the extrinsic pathway using the EXTEM and APTEM tests. Both tests use tissue factor as an agonist with the EXTEM test influenced by extrinsic coagulation factors, platelets, and fibrinogen. In contrast, the APTEM test allows for the investigation of fibrinolysis, which is based on the EXTEM test but with the addition of aprotinin to inhibit fibrinolytic proteins. Fga^-/- mice were bled as described for clot contraction, and the collected blood was reconstituted with recombinant fibrinogen at a final concentration of 0.5 mg/mL (1.47 µM). The assays were repeated three times each. The clot contents of the cups were collected afterwards and fixed overnight in 2.5% glutaraldehyde prepared in 0.9% NaCl (VWR International, Lutterworth, UK). The following morning, clots were washed three times with 0.05 M sodium cacodylate (pH 7.4), dehydrated, critical-point dried, and sputter-coated as described in the clot structure by scanning electron microscopy.

Bovine serum albumin (BSA) and sodium hydroxide were obtained from Sigma–Aldrich (MO, USA). Glutaraldehyde (crosslinker) solution (25%) and Ethanol 96% were provided by VWR BDH Prolabo (VWR, Paris, France). DPBS (+) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Disodium phosphate and monosodium phosphate were provided by Merck GmbH. Electrophoresis gel components, which consisted of acrylamide 40%, Tris electrophoresis purity reagent, sodium dodecyl sulfate, ammonium persulfate, and tetramethylethylenediamine (TEMED), were all provided by Biorad. Ultra-pure water was purified using a synergy unit system (Millipore, Villeurbanne, France).