H e staining kit

Trp53 (1C12), YAP (D8H1X), GAPDH antibodies, and Hippo Signaling Antibody Sampler Kit were purchased from Cell Signaling Technology. Antibody against Rb1 was purchased from Santa Cruz Biotechnology. The secondary fluorescent antibodies and H&E staining kit were from Abcam. DAB Substrate Kit was ordered from Vector Laboratories Inc. Plasmids pRL-TK, 8xGTIIC-luciferase, and shYAP1/2 were obtained from Addgene. The transfection reagents (FuGENE^® HD) were obtained from Promega Corporation.

Sections were stained using a H&E Staining Kit (Abcam, Cambridge, MA, USA), according to the manufacturer's instructions. Morphological changes were evaluated by light microscopy (Nikon Corporation, Japan) at 200x magnification.

The excised tumours were fixed, dehydrated and embedded in paraffin. Then, samples were sectioned at 7 µm thickness and stained by haematoxylin and eosin (H&E Staining Kit; Abcam, Cambridge, UK, ab245880) following the manufacturer's protocols. For immunohistochemistry assay, sections were deparaffinized, hydrated and microwaved for antigen retrieval. After blocking by 5% bovine serum albumin overnight at 4°C, slides were incubated with anti‐Ki67 primary antibody (Abcam, 15580; at 1:200 dilution) overnight at 4°C. Next day, sections were washed three times and incubated with goat anti‐rabbit horseradish peroxidase‐conjugated secondary antibody (Thermo Fisher, 32260; at 1:50 dilution) for 2 hours at room temperature. After diaminobenzidine staining (Sigma‐Aldrich, D12384), images were captured with an optical microscope and analysed using ImageJ v1.53 software.

Cryostat sections were used for histological studies, namely staining with a Hematoxylin and Eosin (H & E) solution, in order to measure the retinal thickness. For this purpose, an H & E staining kit (Abcam, Cambridge, UK, ab245880) was employed, and the detailed protocol followed was the one recommended in the manual of the company.

Lung tissue samples of mice were exposed to 10% formalin and subsequently dehydrated and paraffin‐embedded to prepare into 4‐μm sections. For IHC staining, lung tissue section was incubated with anti‐DNAJB1 (1:50, ab231577, Abcam, Cambridge, MA, USA) and secondary antibodies after dewaxing, inactivation, and sealing using SP Kit (Solarbio, Beijing, China). Then, the sections were treated with DAB solution and hematoxylin followed by detected DNAJB1 positive cells under a microscope. For H&E staining, section was hatched with hematoxylin solution, Bluing Reagent, and Eosin Y Solution using H&E Staining Kit (Abcam). After being washed with absolute alcohol, the pathological changes of mice lung tissue section were observed under a microscope. For TUNEL staining, section was dewaxed, inactivated, and sealed, followed by hatched with Biotin labeling solution Streptavidin‐HRP solution, DAB solution, and hematoxylin using TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China). Then, the apoptosis of mice lung tissue cells was counted under a microscope.

The control group included 81 participants, including 53 women and 38 men, from whom IVDs of the L/S section of the spine were collected post-mortem during forensic autopsy or organ donation. The inclusion and exclusion criteria for the study group are presented in Table 2. They are the same as those shown in previous works [30 (link),31 (link)].
To verify the absence of degenerative changes in the IVD samples, staining was performed using hematoxylin and eosin (H&E) dyes according to the manufacturer’s recommendations (H&E staining kit; Abcam, Cambridge, MA, USA). Two independent investigators evaluated each slide.