Rotation microtome

Mouse eyeballs were enucleated and immediately fixed in 4% paraformaldehyde at 4°C overnight as previously described (Fu et al., 2018b (link)). After rinsed three times with PBS, the eyes were dehydrated and embedded in paraffin (Paraplast, Sigma-Aldrich, USA). Tissue slices (5 μm) were obtained using a rotation microtome (Thermo Fisher, USA), deparaffinized, and then rehydrated with graded ethanol for 5 min twice each. Antigen retrieval was conducted in citrate buffer. Once cooled, tissue sections were blocked with 10% goat serum and 0.2% Triton-X 100 in a dark and humid chamber for 2 h. After rinse briefly with PBS, the sections were immunolabeled with rabbit polyclonal antibody (α-smooth muscle actin, 1:100, Abcam) and incubated at 4°C overnight. After flushing, the samples were incubated with corresponding secondary antibodies (Alexa goat anti-rabbit 568, 1:500, Thermo Fisher) for 2 h. DAPI (Vector, CA) was used to visualize cellular nuclear. The slices were examined by the Keyence all-in-one fluorescence microscope (Itasca, USA) (Kasetti et al., 2016 (link)). For H&E staining, the paraffin section of mice TM tissues were sequentially deparaffinized, rehydrated, stained with hematoxylin and eosin (Sigma-Aldrich), dehydrated and sealed. The slices were visualized and photographed with phase contrast microscope (DMI 1, Leica).

Approximately 5 × 10⁶ H1975-GFP cells were harvested, resuspended in 100 μl PBS, and injected subcutaneously into each mouse's right flank. Treatment was commenced when the tumor size reached approximately 100 mm³. The size of the tumor and the weight of the mice were recorded every day. An animal in vivo imaging system was used to evaluate the tumor's size on Days 7 and 14 after radiotherapy. Tumor volume (V) was calculated according to the formula: π/6 × length × width².
The tissues from the tumor-bearing mice were fixed in 4% PFA at 4 °C overnight and embedded into paraffin (Paraplast, Sigma-Aldrich) using a tissue processor (Thermo Fisher Scientific, Loughborough, UK). Paraffin sections (5 µm) were cut with a rotation microtome (Thermo Fisher Scientific, Bremen, Germany). The images were collected by Versa 8 (Leica, Germany). The integrated optical density of IHC sections was calculated by Image-Pro Plus 6.0.