Immunohistochemistry kit

Continuous sectioned slides (5 μm) obtained from specimens that were previously fixed with 10% formalin solution and embedded with paraffin were stained using an immunohistochemistry kit (ZSGB-BIO, Beijing, China). In brief, xylene and graded ethanol were applied to de-paraffinize the slides, while sodium citrate buffer (0.01 M, pH 6.0) was used to perform antigen retrieval for 20 min at 100℃. All sections were blocked with endogenous peroxidase blockers at 37℃ for 15 min before incubation with primary antibodies at 4℃ overnight. After the reaction with biotin-labeled goat anti-rabbit secondary antibodies for 20 min at 37℃, all sections were washed with phosphate buffer saline (PBS) three times and visualized after staining with diaminobenzidine and hematoxylin. Image-Pro Plus software (version 6.0; Media Cybernetics, Rockville, USA) was applied to measure the mean integrated optical density of each section. Based on the staining intensity and range, the sections were dichotomized into two groups: positive expression (stained brown, and stained tumor cells were no less than 50%) and negative expression (stained yellow, and stained tumor cells were less than 50%).

The rats were sacrificed 2 weeks after the indicated surgery, and IVD tissues were collected. After fixation in 4% paraformaldehyde, the IVD tissues were decalcified, dehydrated, cleared using dimethylbenzene, and embedded in paraffin. Then, they were cut into 5-μm sections. Immunohistochemistry was performed with an Immunohistochemistry Kit (SP-9000, ZSGB-BIO, China) according to the instructions. After dewaxing and hydration, the sections were treated with Tris-ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (C1038, Solarbio) for 10 min at 95°C and endogenous peroxidase blocker for 10 min. After blocking in goat serum for 30 min at room temperature, the cells were incubated with primary antibodies against iNOS (1:200, 18985-1-AP, Proteintech), COX-2 (1:200, ab15191, Abcam), ADAMTS-5 (1:200, ab41037, Abcam), MMP-13 (1:200, sc-515284, Santa Cruz Biotechnology, China), NLRP3 (1:200, Affinity, DF7438) at 4°C overnight. Then, the sections were incubated with goat antirabbit immunoglobulin (IgG) secondary antibodies for 1 h at room temperature and horseradish peroxidase (HRP)-labeled Streptomyces ovalbumin for 15 min. Detection was performed by using the DAB Substrate kit (ZLI-9018, ZSGB). Then, the slides were counterstained with 1% hematoxylin. Images were captured by a microscope (Leica DMI3000B). The positive areas were quantified using ImageJ.

Tissues were isolated and fixed overnight in 4% paraformaldehyde (PFA) and embedded in paraffin. Paraffin sections were subjected to dewaxing, hydration, and antigenic repair (microvaved in sodium citrate antigen repair solution for 20 min), and blocking (5% goat serum in PBS for 1 h). For immunofluorescence, the samples were incubated with primary antibody against Pax6, NeuN, Calbindin, BLBP, and HA overnight at 4 °C, followed by incubation with Alex555- or Alex488-conjugated secondary antibody at room temperature for 1 h. Nuclei were counterstained with 6’-diamidino-2-phenylindole (DAPI), and immunostaining was analyzed by a laser confocal microscope (LSM900). For immunohistochemistry, the samples were incubated with primary antibody against p-p38, Gli1, Ki67, phospho-S937-Gli1, K5, K10 and K17, respectively, overnight at 4 °C, followed by incubation with biotin-labeled goat anti-mouse/rabbit IgG secondary antibody and 3,3’-diaminobenzidine tetrahydrochloride (DAB) developing using an immunohistochemistry kit from ZSGB-BIO (Beijing, China). TUNEL staining was performed following the manufacturer’s protocol using a TUNEL staining kit (Roche, Shanghai, China), and a fluorescence microscope was utilized to examine the TUNEL‐positive cells.

Expression of SCARA5 in paraffin embedded lung cancer, adjacent paracancerous tissues, and xenograft tumor tissues in mice was detected by immunohistochemistry staining. The tissue was cut into 4 μm thick slices, transferred onto glass, and incubated at 65°C for at least 6 h. All the required reagents except antibody came from an Immunohistochemistry Kit (ZSGB-BIO, Beijing, China) and operation was performed according to the standard procedures. Slides were incubated at 4°C for 16–20 h with Ki67 (#16667, Abcam) and SCARA5 (#ab118894, Abcam) antibodies. Then tissues were stained with DAB substrate (K176810E, ZSGB-BIO, China) for 40–50 s. The nuclei were stained with hematoxylin for 5 s and covered with neutral resin. The image of IHC was observed by microscope. IHC scoring criteria: 0: The positive rate was less than 10%; 1: Positive rate was between 10 and 30%; 2: Positive rate was between 30 and 50%; 3: Positive rate was more than 50% added a description of Additional file 2: Table 2.

L-carnitine standard (C0158, CAS 541- 15-1) was purchased from Sigma Aldrich (St. Louis, MO, USA). Primary antibody against p-HSL/HSL, CPT1b and PPARα/UCP1 were purchased from Cell Signaling Technology (Shanghai, China), Bioss (Beijing, China), and Abcam (Shanghai, China), respectively. Antibody against GAPDH and β-actin were purchased from ZSGB-BIO (Beijing, China) and Cell Signaling Technology (Shanghai, China), respectively. Hematoxylin-eosin staining kit was purchased from Beyotime (Beijing, China). Immunohistochemistry kit was purchased from ZSGB-BIO (Beijing, China). FFA measurement kit was purchased from Solarbio (Beijing, China). Other general laboratory supplies were all of the highest grade obtainable.