Ab175388

Immunohistochemistry (IHC) was performed as previously reported [51 (link)]. Anti-SRSF10 (1:200, GTX47232, GeneTex, San Antonio, USA) or anti-CD47 (1:100, ab175388, Abcam) antibodies were used. Each tumor was assigned with a score according to the intensity of the nuclei or cytoplasm staining (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) and the proportion of stained tumor cells (0 = 0%, 1 = 1–10%, 2 = 11–50%, 3 = 51–80%, and 4 = 81–100%), as judged independently by two pathologists in a blinded manner. The final immunoreactive score was determined by multiplying the intensity scores by the extent of positivity scores of stained cells, with “−” for a score of 0, “+” for a score of 1–4, “++” for a score of 5–8 and “+++” for a score of 9–12. Tumors with scores ≥5 were classified into the high expression group, whereas the others were classified into the low expression group.

CD24, CD47, and PD‐L1 expression in tumors were stained by immunohistochemistry using formalin‐fixed paraffin‐embedded tumor tissue specimens. The BenchMark XT used in the immunostaining performed deparaffinization of tissue sections at 75°C using EZ Prep buffer (Ventana Medical System). Antigen retrieval was achieved in Tris‐EDTA base buffer, pH 8.5 (Cell Conditioning 1) for 60 min at 95°C. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 min. The primary CD24 antibody (ab31622; Abcam plc) and CD47 antibody (ab175388; Abcam) were diluted 1:100 and 1:1500 and incubated at 37°C for 60 min. The immunohistochemical reaction was visualized using the Ultraview Universal Diaminobenzidine (DAB) IHC Detection Kit in accordance with the manufacturer's protocol. Patients with known reactivity to antibodies were included as positive controls, and negative controls were obtained by omitting the primary antibody.
CD24, CD47, and PD‐L1 expression in tumor cells were judged to be positive if membranous staining was present and evaluated by TPS, which is the percentage of viable tumor cells showing partial or complete membrane staining. All pathological evaluation was made independently by 2 pathologists (YK and JF) and results were determined by consensus. Both clinical and pathological data were masked at the time of scoring.

IHC was performed on 5-μm sections of tissue specimens following the manufacturer’s protocol. Briefly, tissues were fixed in 4% paraformaldehyde and washed with 10 mM PBS. An isotype-matched monoclonal antibody (mAb) (Iso, anti-rabbit IgG mAb, Equitech-Bio, Kerrville, TX, USA) with no specific affinity to proteins of interest was used as the negative control. Antibodies against CD47 (ab175388, Abcam, Cambridge, UK) and IFN-γ (15365-1-AP, Proteintech, Wuhan, China) as well as the isotype control mAbs were diluted in 5% bovine serum albumin at 25 μg/mL and immobilized on tissue slides at 4°C overnight. For defining the CD47 expression stage, intensity of staining was categorized as 0 (no immunostaining), 1 (weak), 2 (moderate), and 3 (strong); the percentage of chromatists cells as 0 (none), 1 (1%–10%), 2 (11%–50%), 3 (51%–80%) and 4 (>80%). Multiply these 2 numbers: 0–2 is considered (−); 3–4 (+); 5–8 (++); and 9–12 (+++). A range of 0–1+ was considered as negative expression, while 2–3+ was positive expression.⁴⁸ (link) Staining was evaluated by two pathologists in a blinded fashion. The average integrated optical density (IOD) of CD47 and IFN-γ IHC staining in cancer and normal tissue sections were calculated with Image-Pro Plus software (version 5.0, Media Cybernetics, Rockville, MD, USA). Five fields were analyzed per slide.