Forane

Stannous phytate Kits and a Techne Phytate Kit were purchased from FUJIFILM RI Pharma Co., Tokyo, Japan. ^99mTc-phytate solution was prepared by mixing the content of the product with ^99mTc-pertechnetate. Using a 27-gauge needle attached to a 1.0-mL syringe, a dose of 9.3 MBq of ^99mTc-phytate in 0.1 mL was injected into the right side of the tongue in SHO mice under isoflurane inhalation anesthesia (gas anesthesia system for small animals, DS Pharma Biomedical, Osaka, Japan with Forane^®, Abbvie, Tokyo, Japan). Two hours later, the mice were euthanized with an overdose of isoflurane inhalation, and the neck tissue was removed. Autoradiography was performed to detect radioactivity in the lymph nodes of the mice using a phosphorimager (FLA2000; FUJIFILM, Tokyo, Japan) with an imaging plate (IP, BAS-SR2040, FUJIFILM, Tokyo, Japan). The excised neck specimens were placed on the IP covered with a sheet of film of polyvinylidene for 5 min to produce autoradiographic images in a darkroom. Then, the IP was processed to image the radioactivity using the FLA2000 device.

For experimental tumor induction, mice were anaesthetized with a dedicated small animal anaesthesia device (Tec3 Isoflurane Vaporizer, Eickemeyer Veterinary Equipment, UK) using 3% isoflurane (Forane, AbbVie), 0.4 L/min O₂, and 1.4 L/min N₂O. After depilation of the left shoulder area of the animals, CB17 SCID mice (n = 30) were injected subcutaneously with 1.5 × 10⁶ HT1080 (human fibrosarcoma) cells in 150 μL (1/3 part of Matrigel and 2/3 part of saline), and C57BL/6 mice (n = 30) were injected subcutaneously with 3 × 10⁶ B16-F10 cells in 150 μL of sterile saline. Tumor growth was assessed by caliper measurements by the same experienced researcher. The tumor size was calculated using the following formula: (largest diameter × smallest diameter²)/2.

MTLE-HS mice, in which reprogramming of glia had been induced in situ or following grafting, were used for electrophysiological recordings at 2-3 mpi. Mice were deeply anesthetized with isoflurane (Forane, Abbvie), decapitated and the brains were quickly collected into a chilled artificial cerebrospinal fluid (ACSF; Composition in mM: NaCl, 85; Sucrose, 73; KCl, 2.5; NaHCO₃, 25; CaCl₂, 0.5; MgCl₂, 7; NaH₂PO₄, 1.25 and glucose, 10) saturated with 95% O₂ and 5% CO₂ (pH 7.4). Coronal brain slices (250 μm thick) containing the dorsal hippocampus were prepared using a vibratome (VT1200 S, Leica) and transferred to standard ACSF (Composition in mM: NaCl, 125; KCl, 2.5; NaHCO₃, 25; CaCl₂, 2; MgCl₂, 1; NaH₂PO₄, 1.25 and glucose, 12; pH 7.4). Slices were incubated in standard ACSF at 34°C for 1h followed by one additional hour at room temperature (21°C ± 2°C). For recordings, individual slices were transferred into a recording chamber mounted on the stage of an upright microscope (Slice Scope Pro 6000 System, Scientifica, UK). Slices were constantly perfused at the rate of 1-2 mL/min with standard ACSF maintained at 30°C and saturated with 95% O₂ and 5% CO₂. Cells were visualized using a 40X (0.8 numerical aperture) water immersion objective with infrared DIC videomicroscopy.

Immunodeficient CB17 SCID mice (12-week-old; female; n = 35) were housed under sterile conditions in individually ventilated cage system (IVC cages, Techniplast, Akronom Ltd., Budapest, Hungary) under standard conditions (25 ± 2 °C and 55 ± 10%). A sterile semi-synthetic rodent diet (SDS VRF, Animalab Ltd., Budapest, Hungary) and sterile tap water were available ad libitum to all the experimental animals. Experimental animals were kept and treated in accordance with all the corresponding paragraphs of the Hungarian Ethical Laws and the regulations of the European Union (permission number: 16/2020/DEMÁB).
For the establishment of the KB-3-1 cervix carcinoma tumor model, experimental animals were anesthetized by 3% isoflurane (Forane, AbbVie, Budapest, Hungary; OGYI-T-1414/01), O₂ 0.4 L/min and N₂O 1.2 L/min (Linde Healthcare, Budapest, Hungary; OGYI-T-20607 and OGYI-T-21090, respectively) using the Tec3 Isoflurane Vaporizer anesthesia device (Eickemeyer, Sunbury-on-Thames, Surrey, UK), then 5 × 10⁶ KB-3-1 tumor cells in saline (150 µL 0.9% NaCl) were injected subcutaneously into the right shoulder area of the experimental animals. In vivo imaging and ex vivo biodistribution studies were carried out 11 ± 1 days after the implantation of KB-3-1 cancer cells at the tumor volume of approximately 75 mm³.

As part of in vivo imaging 10.03 ± 2.69 MBq of [⁶⁸Ga]Ga-NOTA-c(NGR) and four hours later 11.33 ± 1.71 MBq of 2-[¹⁸F]FDG were intravenously injected into both the ischaemic and the control rats via the lateral tail vein 1, 3, 5, 7, and 10 days post-I/R induction. Sixty minutes post-tracer administration, 20 min static PET images were acquired on the hindlimb region of the rats under isoflurane-induced anaesthesia (3% isoflurane (Forane), AbbVie, Budapest, Hungary; OGYI-T-1414/01) using the MiniPET II device of the Division of Nuclear Medicine and Translational Imaging, Department of Medical Imaging, Faculty of Medicine, University of Debrecen, Hungary.

Female mice at 8–12 weeks of age were anesthetized with 2–4% Isoflurane (Forane, AbbVie Inc.), placed in a stereotaxic frame (RWD Life Science, Shenzhen, China), and kept under constant anesthesia with oxygen at a flow rate of 800 ml/min. Viruses were injected into the ACC (coordinates: anterior–posterior (AP) 1.8 mm, medio‐lateral (ML) 0.5 mm, and dorso‐ventral (DV) 1.25 mm) or CL (coordinates: AP ‐1.2 mm, ML 0.5 mm, and DV 2.7 mm). AAV vectors were delivered to the injection site at a volume of 0.3 μl and a rate of 0.1 μl per minute, using a Hamilton syringe. At the end of the injection, the needle was left in place for 3 additional minutes to prevent the efflux of virus during the removal of the needle. The needle was then slowly removed, and the scalp was sutured. For monosynaptic retrograde tracing, AAV vectors conditionally expressing a cassette for co‐expression of the TVA receptor and the rabies N2c glycoprotein (N2cG) were injected unilaterally in Gal‐Cre, FLEX‐tdTomato, or C57BL/6J female mice, alongside vectors expressing Cre recombinase (for FLEX‐tdTomato and C57BL/6J mice). Two weeks later, pseudotyped rabies viral vectors (RVdG_envA‐CVS‐N2c‐EGFP/tdTomato, ~2–5 × 10⁸ TU/ml) were injected close to the area of the prior injection, and mice were anesthetized and perfused 5–7 days later (Sumser et al, 2022 (link)).

Wild‐type (WT) C57BL/6J female mice (age, 6‐8 weeks) were purchased from SLC (Shizuoka, Japan) and kept in a specific pathogen‐free animal facility at the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN) for at least 1 week before use in experiments. GPR40‐deficient mice have been described previously21 and were bred and maintained in the animal facility at NIBIOHN. For euthanasia, mice were deeply anesthetized using isoflurane (Forane, AbbVie) and then killed through cervical dislocation.