Dab peroxidase substrate kit

Imject^TM Alum Adjuvant (No. 77161) was obtained from Thermo Scientific Inc. (Pittsburgh, PA, USA). Ovalbumin (OVA) was obtained from Sigma-Aldrich Inc. (Saint Louis, MO, USA). BCA protein assay kit was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Goat anti-mouse IgE antibody and HRP-labeled donkey anti-goat IgG antibody were obtained from Abcam Inc. (Cambridge, UK). DAB peroxidase substrate kit was purchased from Solarbio Co., Ltd. (China). The HPLC-grade acetonitrile, methanol, ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water was collected from Millipore Milli-Q system (Bedford, MA, USA). Cellulase (400 U/mg), pectinase (500 U/mg) and papain (800 U/mg) were obtained from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ultrafiltration centrifugal tube (15 mL, 10 kDa) was purchased from Millipore Inc. (Bedford, MA, USA). Other reagents were purchased from Sigma-Aldrich Inc. (Saint Louis, MO, USA).

First, the spleen paraffin sections were deparaffinized in xylene and graded ethanol. To block endogenous peroxidase activity, the sections were incubated with 10% hydrogen peroxide. For antigen retrieval, the sections were heated in 2% EDTA solution for 15 min, and allowed to cool for 2 h at room temperature. After washing with PBS, the slides were blocked with goat serum (Zhongshanjinqiao Biotechnology Co., Ltd., China) for 15 min, and then incubated with the diluted antibodies at 4℃ overnight. On the next day, the sections were incubated with corresponding secondary antibodies at room temperature for 15 min. The sections were then labeled with horseradish peroxidase, and the signals were detected using the DAB Peroxidase Substrate Kit (Solarbio). After staining with hematoxylin (Beyotime) for 30 s, the slides were dehydrated in graded ethanol and xylene. Finally, the sections were sealed with coverslips by resinene (Solarbio). The following primary antibodies were used for the assay: anti-GS (Affinity), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity).

TMA sections of 4 μm thickness were placed on a charged slide, deparaffined, and then rehydrated at a reduced alcohol concentration. Antigens were recovered, sealed, and incubated with primary antibodies. After 6 h, glass slides were treated with secondary antibodies and stained using a DAB peroxidase substrate kit (Solarbio, China).
IHC assays were performed using the following antibodies: CDK1, NUSAP1, CEP55, TOP2A, MELK, PBK, RRM2, and MAD2L1 (Affinity Biosciences, dilution 1:200). Allred scores were used to analyze immunohistochemistry. The positive staining intensity (0: negative; 1: weak; 2: moderate; and 3: strong) was multiplied by the staining area (0: <5%; 1: 5–25%; 2: 26–50%; 3: 51–75%; and 4: >75%). The final score (on a scale of 0 to 12) was converted to a scale from 0 to 3 [a score of 0–1 was considered negative (0); 2–4 was considered weakly positive (1); 5–8 was considered medium (2); and 9–12 was considered highly positive (3)]. After immunohistochemical analysis, the sections were scanned to obtain high-resolution (40X) digital images using a 3DHISTECH scanner (Pannoramic, TaiBei) in the pathology laboratory of West China Hospital.