Tissue processor

The skin tissues that were fixed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) were washed for 30 min for embedding. Skin paraffin blocks made using a tissue processor (Thermo Fisher Scientific, Waltham, MA, USA) were sectioned at 7-µm using a microtome (Leica, Wetzlar, Germany), and cooked at 37°C overnight to keep them attached to the slides. The sectioned slides were passed through xylene and four concentrations of ethanol (100%, 95%, 80%, and 70%) to deparaffinate them for staining.

The skin tissues attached to SAT were fixed with cold 4% paraformaldehyde (Sigma-Aldrich). The fixed tissue samples were washed for 1 h for embedding. Paraffin blocks of skin tissues attached to SAT were then created using a tissue processor (Thermo Fisher Scientific). The paraffin-embedded blocks were sectioned at 7 µm using a microtome (Leica, Wetzlar, Germany) and dried at 60 °C for 24 h to keep them attached to the coated slides.

Collected tissue was fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. Samples were then washed with PBS and placed in an automated Tissue Processor (ThermoFisher Scientific^™, Waltham, MA). Using a ThermoFisher Histostar Tissue Embedding station, processed tissue was imbedded into paraffin blocks. Paraffin blocks were then sectioned at 8μm thickness onto Superfrost/Plus adhesive slides (ThermoFisher Scientific^™, Waltham, MA). Slides were then stained with hematoxylin and eosin (Cat: H-3502; Vector Laboratories, Burlingame, California), Masson’s Trichrome (ab150686; Abcam^®, Waltham, MA), and Picrosirius Red (ab150681; Abcam^®, Waltham, MA). Slides were imaged with a 40x objective using a MoticEAsyScan One slide scanner.

Skin paraffin blocks made by using a tissue processor (Thermo Fisher Scientific, Waltham, MA, USA) were sectioned to 7 µm, using a microtome (Leica, Wetzlar, Germany), and incubated at 37 °C, overnight, to maintain their attachment to the slides. The sectioned slides were passed through xylene and four concentrations of ethanol (i.e., 100%, 95%, 80%, and 70%) to remove the paraffin for staining.
The sectioned skin-tissue slides were incubated in 3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase. The tissue slides were subsequently washed by using PBS and then incubated together with primary antibodies (Supplementary Table S1) in normal serum for 12 h at 4 °C. The slides were rinsed with PBS and then incubated with a biotinylated secondary antibody, using an ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA), for 2 h at room temperature. After washing with PBS, the tissue slides were developed by using 3,3′-diaminobenzidine (Sigma-Aldrich) for 15 min, until confirmation of brown signals. To identify nuclei, the tissue slides were stained in hematoxylin solution for 1 min and then mounted with DPX mounting solution (Sigma-Aldrich). Images of the stained tissues were taken under an optical microscope (Olympus Optical Co., Tokyo, Japan) and analyzed by using ImageJ software (NIH, Bethesda, MD, USA).

The expression of platelet-derived growth factor-C (PDGF-C) in the rat brain’s hippocampus tissue was determined using an immunohistochemistry (IHC) analysis. A total of 2–3 mm thick coronal sections were cut throughout the dorsal hippocampus. Brain sections were processed in a Thermo tissue processor, according to the manufacturer’s protocol for 16 h, this included fixation, dehydration, clearing, and wax infiltration. Brain tissues sections were embedded in molten paraffin wax (Thermo, Waltham, MA, USA) and cooled out; the formed paraffin wax mould was then trimmed for obtaining 3 um sections using a Leica microtome, and the sections were fished out using Poly-Lysin coated slides (Thermo) for IHC staining. All the prepared slides were left at room temperature overnight for dehydration. The IHC-optimized protocol (Abcam) was followed. The primary antibody (Rabbit Polyclonal against PDGF-C, Abbexa) and secondary antibody (Rabbit specific HRP_DAB IHC Detection Kit—Micro-polymer v2d, ab236469, Abcam) were used. The method was performed according to the manufacturer’s (Abcam) instructions. No primary antibody control slide for each sample was run in parallel with the tested slide. The slides were observed under the microscope to score the intensity of staining.

Fixed tissue samples were preceded with a tissue processor (Thermo Fisher Scientific). For hematoxylin and eosin (H&E) stainings, histologic evaluation was performed on 5 μm thick sections and stained with hematoxylin and eosin. High power field images (i.e. 400x magnification) were collected from each colon tissue. For immunofluorescence staining, tissue sections were heated with the antigen retrieval buffer (citrate buffer, pH 6) to 95°C and blocked with 10% goat serum in PBS. Sections were subsequently incubated with rabbit anti-CD3 and bound antibodies were visualized with Alexa Fluor 488 anti-rabbit. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunostained sections were scanned using an Olympus fluorescence microscope. Four representative high power field images were captured from each colon and processed using Olympus cellSens software.

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.