Oct embedding agent

Blood was collected from rats under deep anaesthesia, followed by centrifugating at 4°C for 20 min at 1000x g, and serum was collected. After perfused with saline and 4% Paraformaldehyde Fix Solution (Servicebio, G1101), the rats were executed and brain and heart tissues were collected. The brain tissues were divided into three groups: (1) PVN was carefully removed directly from both sides of the third ventricle; (2) brain tissue was frozen after being covered with OCT embedding agent (Servicebio, G6059); and (3) brain tissue was embedded in paraffin. The heart tissues were divided into two groups: (1) 3 mm of myocardial tissue surrounding the infarct region of the left ventricle in rats was carefully removed, and (2) the whole heart tissue was embedded in paraffin.

Fresh tumor tissues were fixed and dehydrated as has been proposed. For mIF, tumor sections from the following independent ICI cohort (2 patients for MPR, Non, and pre respectively) were collected. OCT embedding agent (Servicebio) was used to embed tissues before staining. Briefly, 3% BSA (diluted in PBS) was adopted for blocking non-specific binding, then anti-human ADGRE5 Ab (1:150) (Absin, Cat: abs132702) was incubated at 4°C overnight. PBS (pH 7.4) washing of slides 3 times was conducted. Slides were then incubated with Cy5-goat anti-rabbit (1:300) for 70 min in dark (room temperature). After washing by PBS, anti-human CD8α Ab (1:300, Absin, Co: abs158658) staining was performed. Then counterstain of the DAPI solution (1:4000) was performed. Leica Sp8 laser scanning confocal microscope and ImageJ software were used for imaging and image-data processing respectively.