P midi

Immunohistochemistry was used to determine the FTO expression level in EC tissue samples. The methods followed have been described in detail in a paper published by our research group [17 (link)]. Anti-FTO (1:500, Abcam) was used as the primary antibody. The slices were then imaged using a professional microscope (P-MIDI, 3D HISTECH, Hungary).

Sections were fixed using 4% paraformaldehyde and blocked with 10% goat serum at 37°C. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 h at 37°C following the day. TSA was incubated at 37°C for 30 min. After washing with PBS, the fixation step, serum blocking, and antibody incubation were repeated. After final fixation, the cells were stained with DAPI. Anti-fluorescence quencher containing Hoechst33342 (P0133, Biyuntian) was used to seal tablets. The primary antibodies were rabbit anti-LRP1 (1:200, ab92544; Abcam), rabbit anti-RAGE (1:200, ab3611; Abcam), and rabbit anti-CD31 (1:2,000, ab182981; Abcam). The second antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:300, 074-1506, KPL) and HRP-conjugated goat anti-mouse IgG (1:300, 074-1806, KPL). Photographs were taken using a Pannoramic DESK, P-MIDI (3D HISTECH, Hungary) scanner. The percentage of positive areas was analyzed using ImageJ software. ImageJ was used to analyze LRP1-CD31 co-localization area, CD31 expression area, RAGE-CD31 co-localization area, and CD31 expression area in the hippocampus and cortex, respectively, and the ratio of co-localization area to CD31 area represents the relative amount of positive protein expressed per unit endothelial cell.

Sixty-eight samples were obtained from patients who suffered from hepatocellular carcinoma and underwent hepatectomy in The First Affiliated Hospital, Zhejiang University School of Medicine. Paraffin-embedded tissue was sliced and dewaxed. After antigen retrieval, primary antibodies were incubated with slides at 4°C overnight. Herein, CD138 (rabbit monoclonal to Syndecan-1/CD138, Cat. No. ab128936, Abcam, United Kingdom), CD86 (rabbit polyclonal to CD86, Cat No. DF6332, Affinity Biosciences, USA), and PD-L1 (mouse monoclonal to PD-L1/CD274, Cat No. 66248-1-Ig, Proteintech, USA) were used. After washing, secondary antibodies were incubated at 37°C for 30 min and washed. Then diaminobenzidine (DAB) was applied for color development. Lastly, all sections were scanned by Pannoramic DESK, P-MIDI, P250, P1000 (3D HISTECH; Hungary) and were read by Pannoramic Scanner (3D HISTECH; Hungary).
All sections were evaluated by three pathologists independently. CD138 and CD86 were categorized into positive (+) or negative (−), and PD-L1 was scored ranging from 0 to 3 (0, 1, 2, and 3). If there was disagreement of the evaluation, the results of CD138 and CD86 adhered the principal of minority obeying majority, and the final score of PD-L1 was the average of values from three observers. The score of PD-L1 was categorized into two groups (high and low) according to the median value.