Imagej pro

Paraffin-embedded tissue sections were dewaxed (xylene, graded ethanol), peroxidase activity quenched (0.3% hydrogen peroxide), and antigen-retrieved; The U87 and U251 glioma cells were fixed in 4% paraformaldehyde. Then they were further blocked with 5% goat serum incubated with primary antibodies (Notch1:100, Abcam, ab52301; Notch2: 1:50, Santa Cruz Biotechnology, sc-518169; Notch3 1:50, Santa Cruz Biotechnology, sc-518169; Notch4 1:50 dilutions, Santa Cruz Biotechnology, sc-32613, Ki67, abcam15580) at 4°C overnight. Subsequently, specific secondary antibodies were used to incubate the sections or glioma cells for 1 hour at 37°C. Then the sections were immersed in ABC peroxidase with diaminobenzidine (Beyotime, China) and counterstained with Mayer hematoxylin for 2 min (Beyotime, China). The U87 and U251 glioma cells were stained and covered with a DAPI solution. A microscope (Nikon, Tokyo, Japan) was used to photograph the staining signals. ImageJ Pro (Media Cybernetics, Rockville, Maryland, United States) was used by two observers (blinded to the experimental groupings) for statistical analysis.

The sections were dewaxed, antigen-retrieved, and blocked. Then, specific antibodies (anti-HOXAs: HOXA1: ab230513; HOXA2: ab229960; HOXA3: ab230879; HOXA10: ab191470, Abcam) covered the sections at 4 °C overnight. After washing three times with PBS, specific secondary antibodies covered the sections for 1 h at 37 °C with horseradish peroxidase, then immersed in diaminobenzidine (DAB) and counterstained with hematoxylin for 2 min. A microscope (BX50/BX-FLA/DP70, OLYMPUS) was used to observe the staining signals. ImageJ Pro (Media Cybernetics, USA) was used by a technician (blinded to the experimental groupings) to quantify the integrated optical densities.