2.3. Immunohistochemistry and evaluation of ATAD3A protein expression

The expression of ATAD3A was detected by immunohistochemistry. During the experiment, 4 mm sections were cut from each sample tissue block and fixed on the slide. And then put them into an oven at 60°C for 2 h. After that, the sections were taken out and dewaxed in xylene, followed by hydration based on gradient ethanol solution and microwave heating in 10 mm citric acid for 10 min. Put the slices treated above into 3% hydrogen peroxide for 10 min to inactivate them. And 1% bovine serum albumin was added. After that, the sections were incubated with anti‐ATAD3A mouse monoclonal antibody (Santa Avenue, CA, dilution range 1:50–1:500) for 16 h at 4°C. The sections were taken out and fully washed, and then treated with secondary antibody (envision, dilution range 1:200–1:500). The sections were refixed with hematoxylin and stored as required after dehydration.

The immunostaining score was distributed independently by two individuals who did not have access to the pathological and clinical data. The score was based on the number of stained cells. If there was a difference between the observers, a consensus score was selected for evaluation. The staining intensity was graded based on criteria: to no staining, light yellow, brownish yellow, and brownish‐yellow staining correspond to 0, 1, and 2, 3 points, respectively. Less than 5% of the stained cells were assigned 0 points, 6%–26% of the stained cells were assigned 1 point, 26%–50% of the stained cells were assigned 2 points, and 50% or more cells stained were assigned 3 points. Based on multiplying positive cells score by the score of the staining intensity, the final score was got: 0–4 was considered a low level of ATAD3A expression, and 5–9 was considered a high level of ATAD3A expression.