Picric acid

Surgery was performed in anaesthetized rats using sodium ketamine (100 mg/kg) and xylazine (Rompun 2%; 10 mg/kg), given intraperitoneally (i.p.). After laparotomy, Fluorogold (FG, Hydroxystilbamidine, methanesulfonate, 4%, 1 µl, 10 injections; Interchim, Montluçon, France) and Fluororuby (FR, Dextran, Tetramethylrhodamine, 10000 MW, Lysine Fixable, 10%, 1 µl, 10 injections; Interchim, Montluçon, France) (17 (link)) were injected in the all part of the urinary bladder wall and in the wall of the colorectum segment respectively. Seven days after these injections, the L6 and S1 DRGs were extracted after intracardiac perfusion using Tyrode solution (NaCl 6.8 g; KCl 0.4 g; MgCl₂ × 6 H₂O 0.15 g; MgSo4 × 7H₂O 0.1 g; NaH₂PO₄ 2H₂O 0.19 g; Glucose 1 g; NaHCO₃ 2.2 g; Fill up to 1l with H₂O), followed by a fixation solution composed of 4% paraformaldehyde (Sigma) and 0.3% picric acid (Sigma) in 300 ml of 0.1 M phosphate buffer. After overnight incubation in fixation solution, DRGs were further incubated in sucrose (10%, 20% and 30%) at 4°C during 24 h each one. This was followed by inclusion in TissueTek®, freezing in isopentan at −40°C, and cryostat sectioning after 24 h at −80°C (LEICACM1950, Ruel-Malmaison, France; 20 µm). A fluorescent microscope was used for identification of colorectum-only, urinary bladder-only and colorectum-urinary bladder co-innervating DRG neurons.

TNG, EGDN, PETN standards were purchased from HPC (Cunnersdorf, Deutschland), HMX, TNT, RDX and Tetryl purchased from Ultra Scientific (North Kingstown, USA) and picric acid and DNT others were purchased from Sigma-Aldrich and Supelco (Germany). Other chemicals and solvents used in the analysis were chromatographic purity and purchased from Merck, Fluka, Sigma-Aldrich, Supelco, and LabScan companies. Standards and real explosive sample including TNT and RDX seized from terrorists were solved in water-ACN (60%–40%) and filtered by 0.45 μm PTFE syringe tip filter. The sample and standard mix solutions were directly injected into the HPLC system.

PEA, C16:0; SEA, C18:0; OEA, C18:1; AEA, C20:4; LEA, C18:2; LNEA, C18:3; EPEA, C20:5; d₄-SEA; and d₄-LNEA were synthesized and completely characterized in our laboratories as previously described [29 (link),30 (link),31 (link),32 (link)]. HPLC grade and LC-MS grade organic solvents were purchased from Panreac Quimica Sau (Barcelona, Spain). Acetonitrile (HPLC grade) and formic acid (used for elution of the analytes), NaOH, and picric acid were purchased from Sigma-Aldrich (Munich, Germany). Solid-phase extraction cartridges OASIS HBL (30 mg/mL) were purchased from Waters (Etten-Leur, The Netherlands).

WVP peptide was manufactured using the solid-phase peptide synthesis method by ChinaPeptides Co., Ltd. (Shanghai, China). During the synthesis process, the C-terminus of WVP was modified with DOTA to obtain DOTA-WVP. Sodium acetate (NaOAc), hydrogen chloride (HCl), 3-aminopropionitrile fumarate salt (BAPN), Sirius red, and picric acid were supplied by Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-mouse Col-IV and elastin were purchased from Abcam, Inc. (Cambridge, UK). Other chemicals and solvents were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

To trace the viability and distribution of the implanted HUMSCs, the cells were labeled with 1 g/ml bisbenzamide (Sigma B2883) for 48 h before trypsination and transplantation. Following sacrifice, mice were perfused with the fixative of 4% paraformaldhyde (Merck Un2213) and 7.5% picric acid (Sigma 925–40) in 0.1 M phosphate buffer (PB). The brain tissues were post-fixed in the same fixative for 24 h and cryoprotected in 30% sucrose in 0.1 M PB. Following sedimentation, the tissue was embedded in optimum cutting temperature compound, frozen, and cryo-sectioned at 30 μm in a cryostat (− 20 °C). Frozen sections were thaw-mounted onto coated glass slides, and directly observed and photographed under fluorescence microscope. In addition, immunostaining using anti-human specific nuclear antigen was performed to trace the survival of HUMSCs. Tissue sections were first reacted with a primary antibody (mouse anti-human-specific nuclear antigen, 1:100, Chemicon MAB1281, 1:100) at 4 °C for 18 h, washed with 0.1 M PBS, reacted with secondary antibodies (biotin-conjugated goat anti-mouse-IgG, 1:300 diluted, Sigma) at room temperature for 1 h. After the chromogenic reaction, the sections were coverslipped and observed under a microscope.

To screen for HCN production, strain FG106 was cultured on glycine-containing King’s B medium that was then pressed against filter paper impregnated with picric acid (Sigma-Aldrich, Taufkirchen, Germany) [51 (link),52 (link)]. The siderophore assay was performed using Chrome Azurol S medium (Sigma-Aldrich, Taufkirchen, Germany) containing iron chloride [52 (link),53 (link)]. Biofilm formation was determined in 96 -ell polystyrene microtiter plates (Nunc^™ MicroWell^™ 96-well and flat- bottom microplate, Thermo Fisher Scientific, Waltham, MA, USA) [54 (link)].

Female (n = 12, 3 for each group) Balb/C intact mice supplied by Animal Care and Usage Comittee of Akdeniz University were maintained under standard laboratory conditions (21 ± 1°C; ambient temperature; controlled light/dark conditions, 14 L: 10D) and were given food and water ad libitum. The day of birth was designated as PND0 and the experimental protocol was approved by the Animal Ethics Committee of Akdeniz University, Turkey (2009.08.36).
Ovaries were harvested from postnatal days (PND) 1, 7, 21 and 60 female mice and left ovary samples were fixed by immersion in Bouin’s fixative (75 mL of saturated aqueous solution of picric acid [Sigma-Aldrich Co. LLC, Steinheim, Germany], 25 mL of formalin [Merck, NJ, USA] and 5 mL of glacial acetic acid [Sigma-Aldrich Co. LLC, Steinheim, Germany]) at room temperature for 4 hours. Then tissues were dehydrated through a graded series of ethanol, cleared with xylene and finally embedded in paraffin wax for immunohistochemical investigations. Right ovary samples were preserved in -80°C freezer until they were processed for protein extraction for Western blotting.