2 solution dab kit

Immunohistochemistry staining was performed as protocol previously described [21] . Briefly, after fixation with formalin, tissues were embedded with paraffin, cut, and mounted on slides. Then, slides were washed with xylene to deparaffinize, with graded ethanol to rehydrate, incubated with citrate buffer to retrieve antigen, and blocked with 3% H₂O₂ to inactivate endogenous peroxidase. Slides were incubated with E2A primary antibody (Santa Cruz Biotechnology, Texas, USA; 1∶200 dilution) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Santa Cruz) for 30 minutes at room temperature. Complex visualization was done with a 2-Solution DAB Kit (Invitrogen). Negative controls were obtained by replacing the E2A primary antibody with preimmune rabbit serum.
Slides were examined by two researchers (Huang and Quan) independently. Scoring criteria used were as previously described with minor modifications. Staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining); positive cells on each section were scored as 0 (less than 10%), 1 (10%-25%), 2 (26%–50%), and 3 (>50%). The final score was a product of scores of intensity and positive cell of each slide. Slides with score 0–3 were defined as low expression and 4–9 as high expression.

Fresh, matched CRC tissues and adjacent normal tissues used for biological analyses and organoid establishment were obtained from Jiangsu Province Hospital of Chinese Medicine (Nanjing, China). All patients gave written informed consent for participation in this study. The study on fresh clinical samples was approved by the ethics committee of Jiangsu Province Hospital of Chinese Medicine.
IHC was performed by Nanjing Microworld Biotechnology Co., Ltd. using paraffin-embedded, formalin-fixed tumor tissues removed from the tumor-bearing nude mice at the end of the study. Sections were incubated with antibodies specific for PRMT1 (Proteintech, 11279–1, 1:100), H4R3me2a (PTMBio, PTM667, 1:100), EGFR (Abcam, ab52894, 1:200), TNS4 (Abcam, ab99887, 1:200) and SMARCA4 (Abcam, ab110641, 1:100). Immunocomplexes were visualized using a 2-Solution DAB Kit (Invitrogen).

IHC was performed on paraffin-embedded sections according to standard protocols. Briefly, tissues were fixed in 4% paraformaldehyde solution or 10% neutral-buffered formalin at room temperature overnight and paraffin-embedded following standard procedures. The sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol (100, 90, 80, 75%) for 3 min each time and microwaved-heated in sodium citrate buffer for antigen retrieval. Then, the sections were blocked in 5% BSA and incubated with primary antibodies: anti-LDHA (1:100 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA #3582), anti-CD33 (1:100 dilution, ProteinTech Group, Inc., Wuhan, China, #17425), anti-LOX-1 (1:200 dilution, ProteinTech Group, Inc., #11837), or anti-c-Rel (1:100 dilution, Santa Cruz Biotechnology, USA, #sc-6955), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Antibody binding was visualized using a 2-Solution DAB Kit (Invitrogen). The images were obtained with an inverted microscope (Olympus IX71, Japan). An H-score was calculated using the following formula: H-score = ∑ (PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + percentage of cells of strong intensity × 3). Here H-score was recorded as a continuous variable.