Xylazine

The drug response trial was separated into two portions. Heifer B was used in the first portion and Heifer A was used in the second portion (Table 1). Infrared images were taken from the right flank, the coronary band, the foot, and the hind area (Fig. 1; ak, g, d, co). In the first portion, where only xylazine (Rompun, Bayer) was utilized, baseline images were taken every 7.5 min for three sets with the FLIR SC2000 camera at a distance of 0.8 m. The animal was then injected with 0.02 mg/kg of xylazine (Rompun, Bayer) intravenously to obtain mild sedation while maintained in the calorimetric chamber, and a subsequent set of images were taken after this injection every 7.5 min for a total of 12 sets. The second portion of the trial involved xylazine and atipamezole (Antisedan, Pfizer), with xylazine first administered to the animal (0.02 mg/kg; IV), followed by atipamezole (0.10 mg/kg; IV) 40 min after the xylazine. A set of baseline images was taken every 20 min for three sets, followed by the xylazine injection. A set of images was then taken every 20 min following the xylazine injection for two more sets, and after injection of atipamezole, two additional sets of images were taken every 20 min. Calorimetry measures were taken for the animals throughout the trial, with information gathered for heat production.

Before each vaccine injection, mice were anesthetized with ±150 μl of a solution of 10 mg/ml ketamine (Ketalar, Pfizer, New York) and 1 mg/ml xylazine (Sigma, St. Louis, Missouri). The left paw was shaved using a rodent shaver (Aesculap Exacta shaver, AgnTho’s, Sweden). Mice were injected with 1 μg of every plasmid (pDNA), or with 1 μg of pTOP-OVA CD4-OVA CD8 irrelevant plasmid, diluted in 30 μl of PBS in the left tibialis cranial muscle. The paw was then placed between 4 mm plate caliper electrodes (BEX Co., Ltd., Japan) and electroporated (200 V/cm, 8 pulses, 20 ms with 500 ms pause between pulses). The pulses were delivered by a CUY21EX electroporator (BEX Co., Ltd., Japan). The vaccine was administered 2, 9 and 16 days after tumor injection (Fig. 1a).

The method for in situ imaging was established in the Zipfel group (Aum et al., 2017 (link)). Mice were anaesthetized using a ketamine (Bayer, Reading, UK) and xylazine (Pfizer, Tadworth, Surrey, UK) mixture (100 and 10 mg/kg, respectively) and transcardially perfused through the left ventricle with 10 mM glucose–physiological-buffered saline (PBS) followed by 20 ml 20 μM 5-(6)-carboxy-X-rhodamine, succinimidyl ester (Sigma-Aldrich, Gillingham, Dorset, UK) dye in 10 mM glucose-PBS prior to perfusions with 4% paraformaldehyde (in PBS). All perfusions were performed with solutions at 21°C, at a constant pressure of 80 ± 2 mmHg using a GE Druck DP1705. Animals were decapitated, the calvaria removed and post-fixed in 4% paraformaldehyde in the dark at 4°C for 24 h. Brains were removed under a dissection microscope to preserve the basal arteries. Then, brains were placed en bloc on a glass coverslip and gently covered in PBS before placing on the stage of a confocal laser scanning microscope (SP8, Leica, Wetzlar, Germany). Measurements of anterior and middle cerebral artery (MCA) diameters were made at the narrowest point across the first millimetre of the vessel, as detailed in Supplementary material.