Graded alcohols

The tissue microarray containing 27 samples of NB patients and 5 peripheral nerve tissues was purchased from Biomax (MC642, Derwood, USA). NB xenograft tumor tissues and mouse organ tissues obtained from animal experiments were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) and embedded in paraffin. Sections were deparaffinized in xylene (MACKLIN, Shanghai, China), rehydrated in graded alcohols (Sinopharm, Beijing, China), and washed in distilled water. Antigens were retrieved by boiling the sections in citric acid-based antigen unmasking solution (Thermo Fisher, Waltham, MA, US) for 30 min, then incubated with primary anti-BRD4 (1:200, ab128874, Abcam, Cambridge, UK) or Ki-67 (1:200, ab15580, Abcam, Cambridge, UK) overnight at 4 °C followed by rabbit specific HRP/DAB detection kit (Cat: ab64261, Abcam, Cambridge, UK) and hematoxylin (Beyotime, Shanghai, China) in compliance with protocols. Staining results were independently evaluated by two experienced pathologists. The total scoring (TS) results were scored by multiplying the percentage of positive cells (P) by the intensity (I). Formula: TS = P × I.

IHC was performed using standard techniques. Briefly, 5-µm paraffin-embedded TMA sections were dewaxed using xylene (Sinopharm, China)and rehydrated in graded alcohols (Sinopharm, China). Next, blocked endogenous peroxidase with 3% hydrogen peroxide (sinopharm, China). Antigen retrieval was accomplished via adding 10 mM citrate buffer (pH 6.0) (Sinopharm, China) to the TMA sections and putting them into a high-pressure cooker. Then, the TMA sections were incubated with 1% bovine serum albumin (BSA; Sigma, German) for 20 min to reduce nonspecific protein binding. The recombinant rabbit monoclonal CDC45 antibody (1:50; HuaBio, Hangzhou, China) were next used to treat the TMA sections at room temperature for 1 h. They were then rinsed with phosphate-buffered saline (PBS; HuaBio, Hangzhou, China) and incubated with biotinylated secondary antibody (MXB, Fuzhou, China) for 30 min at room temperature. Subsequently, the TMA sections were stained with DAB chromogen (Gene Tech, Shanghai, China), counterstained with Mayer’s haematoxylin (HuaBio, Hangzhou, China), dehydrated with gradient alcohol and xylene, and mounted on slides. Finally, the TMA sections were viewed under a Nikon light microscope.