Immunohistochemistry (IHC) analysis was performed as described (Wang et al., 2022 (link)). In brief, the paraffin-embedded sections were deparaffinized and hydrated. Antigen retrieval was achieved using Tris-EDTA buffer at pH 9.0. After endogenous peroxidase activity was blocked, the sections were incubated with an anti-AKR1B10 antibody (cat#ab192865, Abcam, Cambridge, United Kingdom) at a dilution of 1:400 at 4°C overnight. Primary rabbit IgG antibody (cat#ab172730, Abcam, Cambridge, United Kingdom) was used as the negative control. Staining was visualized by using 3,3′-diaminobenzidine tetrahydrochloride (cat#SP-9000, ZSBio, Beijing, China) and hematoxylin counterstain. Representative images were captured under a light microscope (Leica, DMIL LED, Wetzlar, Germany; magnification, ×200). The intensity of IHC staining was designated 0 for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. AKR1B10 immunostaining was based on the positive cytoplasmic staining and was quantitatively assessed as the average percentage of AKR1B10-positive areas in three independent fields. The extent of stained areas was determined as the percentage of stained areas, ranging from 0% to 100%. The IHC score was obtained by multiplying intensity scores with proportion scores, as described (Qu et al., 2018 (link)).
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