Sp 9002

After dewaxing, the paraffin sections were subjected to antigen retrieval with citrate buffer (pH = 6). Then, sections were rinsed with phosphate buffer saline (PBS, Beyotime, Shanghai, China) thrice and blocked with goat serum (SP9002, ZSGB-BIO, Beijing, China) for 20 min at the room temperature. Next, sections were incubated with CD68, Iba-1, C3, and the GFAP antibody overnight at 4°C. Subsequently, sections were washed with PBS three times and incubated with FITC conjugated Goat anti-rabbit IgG (H + L) (1:100, GB22303, Servicebio, Wuhan, China) or Cy3-conjugated Goat anti-rabbit IgG (H + L) (1:100, GB21303, Servicebio) at 37 °C for 30 min. The nuclei were stained with DAPI (ZLI-9557, ZSGB-BIO) for 10 min at room temperature. Images were captured with a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).

After dewaxing, paraffin sections were treated with citrate buffer (pH = 6) to retrieve antigens. Then, sections were rinsed with phosphate buffer saline (PBS; Beyotime, Shanghai, China) thrice and blocked with goat serum (SP9002; ZSGB-BIO, Beijing, China) for 20 min at room temperature. Next, the sections were incubated with the iNOS antibody (1 : 100, ab178945; Abcam, Cambridge, UK), CD206 antibody (1 : 100, 24595; Cell Signaling Technology, Inc., Danvers, MA, USA), HLA-DR antibody (1 : 100, MA5-32232; Thermo Fisher Scientific, Waltham, MA, USA), CD11c antibody (1 : 100, A1508; ABclonal, Wuhan, China), ICAM-1 antibody (1 : 100, 10020-1-AP; Protein Tech, Wuhan, China), and VCAM-1 antibody (1 : 100, ab134047; Abcam) overnight at 4°C. Subsequently, the sections were washed with PBS three times and incubated with FITC-conjugated Goat Anti-Rabbit IgG (H + L) (1 : 100, GB22303; Servicebio, Wuhan, China) or Cy3-conjugated Goat Anti-Rabbit IgG (H + L) (1 : 100, GB21303; Servicebio) at 37°C for 30 min. The nuclei were stained with DAPI (ZLI-9557; ZSGB-BIO) for 10 min at room temperature. Images were captured using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).

Pathologists were asked to confirm the tumor and nontumor tissue samples with hematoxylin‐ and eosin‐stained sections. Paraffin‐embedded tumor and adjacent normal tissue samples were sectioned (4 μm thick). After being dewaxed and rehydrated, an immunohistochemistry kit (ZSGB‐BIO, SP‐9001, SP‐9002) was then used for immunohistochemical (IHC) staining. Following these steps, incubation with methanol containing 0.3% hydrogen peroxide was performed for 30 minutes to block endogenous peroxidase activity. The sections were heated in a pressure cooker filled with the Sodium Citrate Antigen Retrieval solution (Solarbio, C1032) and incubated for 2.5 minutes after boiling with obvious steam. Natural cooling was performed, and the sections were and incubated for 30 minutes in 1% blocking serum to prevent nonspecific binding. Primary antibodies specific for Ubqln2 (Abcam, ab190283) and Ki‐67 (BD Biosciences, 550609) were diluted 1:200 and incubated with the tissue sections at 4°C overnight. Incubation with a biotinylated secondary antibody was performed for 30 minutes. Incubation with a streptavidin‐biotin complex conjugated with horseradish peroxidase was performed for 30 minutes. Diaminobenzidine (ZSGB‐BIO, ZLI‐9018) was used to develop the visualization signal. The sections were counterstained with hematoxylin.

Tissue specimens were obtained from the tumour xenograft experiment mentioned above. The paraffin-embedded tissues were dewaxed, rehydrated, and stained using the Immunohistochemistry kit according to the manufacturer’s protocol (SP-9002, ZSGB-BIO, China). The antibody used for IHC was anti-4-HNE (1:200, Abcam, ab46545) and ODC1 (1:100, A3898, Abclonal).

For immunohistochemistry (IHC), patient tissues were fixed in 4% Paraformaldehyde, embedded in paraffin, and then cut into 5 μm thick sections for IHC staining. The slides were blocked with non-immune goat serum and incubated with anti-mouse GFRA1 antibody (1:200 Santacruz #sc-271546) for 24 h at 4°C. Then follow the instructions (ZSGB-BIO #SP-9002, Beijing, China).
For immunofluorescence, the transfected HCT116 cells and SW480 cells grown on the cover glass in 24 well plates were fixed with 4% paraformaldehyde (PFA) for 20 min and permeabilized with 0.2% Triton X-100 for 15 min at room temperature (22–25°C). Then, the cells were incubated with the desired primary antibodies in PBS for 24 h at 4°C, washed with PBS 3 times, and then incubated with fluorescent secondary antibodies away from light at room temperature (22–25°C). After that, the nuclei were stained with DAPI. The fluorescence intensity and absorbance were measured at 555 nm (Invitrogen #A21428, MA, USA) and 488 nm (Invitrogen #A11001, MA, USA), respectively.

Paraffin-embedded tissue sections (4.0 µm) were deparaffinized, dehydrated, and rehydrated in a graded ethanol series. Three percent H₂O₂ was added to block endogenous peroxidase activity. After a rinse in distilled water and phosphate-buffered saline (PBS) successively, sections were placed in citrate buffer (10 mM/pH 6.0) and heated in a microwave oven at 95 °C for 30 min. Then, the sections were incubated with primary antibody (Anti-FLJ20294, 1:200, Abclonal) at room temperature for 1.5 h and 4 °C over-night. The color reaction was developed with the HRP-linked polymer detection system (SP9002, ZSGB-BIO, Beijing, China) and counterstaining with hematoxylin. Images were captured with an Axio Scope A1 microscope (Carl Zeiss, Germany) and analyzed using the ZEN Blue Lite software (Carl Zeiss, Germany).