Ts 100c

All reagents and equipment used in these analyses were from Biorad (Hercules, NJ, USA). Samples were prepared by mixing 50 mg of WT with 2 mL of 0.1 M sodium phosphate buffer pH 8 (10 min, 1000 rpm (TS-100C, Biosan, Riga, Latvia)), then centrifuged for collection of the supernatant (5 min 10,000 rpm (Ole Dich, Hvidovre, Denmark)). An amount of 10 µL of the supernatant was then mixed together with 5 µL 4× Laemmli buffer, 4.75 µL of milliQ water and 0.25 µL of β-mercaptoethanol. The solution was then incubated for 10 min at 95 °C in a TS-100C heating block (Biosan, Latvia, Riga). An amount of 10 µL of the incubated sample was then loaded into a well in a 4–20% Mini-PROTEAN^® TGX gel where the first well was loaded with 5 µL Precision Plus Standard ladder. The gel was run at 140 V, 400 mA for 50 min. The gel was then washed and stained for 1 h (Coomassie R-250) followed by a de-staining procedure. This consisted of replacing the staining reagent with water and leaving the gel with gentle shaking for 1 h, before replacing the water with fresh water. The last procedure was repeated 3 times before scanning the gel using a ChemiDoc XRS + System.

Briefly, 1.8–14.4. nmol of BCAs or peptide was sequentially loaded into a 0.5 mL Eppendorf-type PP tube. Next, 25 µL of 0.1–1 M aqueous solution of BEA salts 1–16, 37.5 µL of acetone and 125 µL of ⁶⁸GaCl₃ solution in 0.1 M HCl were sequentially added to the test tube. The reaction was carried out by stirring in a thermo-shaker (TS-100C, Biosan, Latvia) for 10–30 min and heating to 37 °C. After cooling, RCCs were measured by radio-TLC (iTLC-SG strips; eluent—50% acetonitrile in water; R_f 0.6–0.8 for radiolabeled peptides and 0.0 for [⁶⁸Ga]Ga³⁺ and ⁶⁸Ga in colloidal form).

Twenty male rats weighing 450–500 g were individually housed in plastic cages, maintained on a 12:12 h light-dark cycle, and fed ad libitum. All animals were decapitated under anesthesia with ketamine and xylazine in a dose mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), in order to collect the brain samples. The brains were rapidly removed, immediately frozen in liquid nitrogen, and stored at −80 °C until analysis. For MDA analysis, brains were homogenized in IKA Ultra-Turrax Tube Drive and were subsequently divided in equal quantities. Afterward, 1 g of brain sample was spiked with 10 μL of working solution, and then PBS was added in a three times higher volume than the sample volume. Samples were vigorously vortexed for 1 min, and immediately after, samples were centrifuged (10,000× g for 10 min). After centrifugation, acetonitrile (ACN) was added for protein precipitation (1:3, v/v). The samples were centrifuged (10,000× g for 10 min) and the collected supernatant was diluted with pure water (1:1, v/v). A volume of 600 μL TBA (4 mg/mL) and 1000 μL sulfuric acid (2.66 μL/mL) were added to 400 μL sample, followed by heating at 100 °C for 60 min in TS-100C, Thermo-Shaker (BioSan, Riga, Latvia). After heating, the samples were transferred in HPLC vials and analyzed shortly after the derivatization reaction.