Edta antigen retrieval buffer

Paraffin sections from TMA including 80 benign ovarian cyst tissues and 115 OC tissues, and murine ovarian cancer tissues were deparaffinized with xylene, anhydrous ethanol and alcohol. Subsequently, they were washed with distilled water. Following that, the sections underwent antigen retrieval using the EDTA antigen retrieval buffer (Servicebio, #G1203, China). Afterwards, endogenous peroxidase activity of the sections was inhibited with 3% hydrogen peroxide solution. Subsequently, the sections were blocked with 3% BSA. Then, corresponding primary antibodies (refer to Additional file 1: Table S1) were applied and incubated overnight at 4 ℃.Thereafter, the related secondary antibodies were incubated, followed by applying a DAB substrate kit (DAKO, #K5007, Denmark).Then, the nuclei were counter stained. After that, the sections were dehydrated and mounted, and observed through a microscope (CIC, XSP-C204, China).

Conforming to the standard protocol, immunofluorescence staining was executed using the following antibodies and dilutions: TNF-α (1:500; Servicebio, Wuhan, Hubei, China), IL-6 (1:500; Servicebio, Wuhan, Hubei, China), and F4/80 (1: 1,000; Servicebio, Wuhan, Hubei, China). After being deparaffinized and rehydrated, the samples were subjected to epitope retrieval by microwaving the slides in an EDTA antigen retrieval buffer with a pH value of 8.0 (Servicebio, Wuhan, Hubei, China). The samples were first permeabilized with BioDewax and Clear Solution (Servicebio, Wuhan, China) and blocked with 3% BSA. Only one antigen was found in each stage, which included primary and secondary antibody incubation, as well as tyramine signal amplification (TSA) visualization. After protein blocking and epitope retrieval as mentioned prior, the next antibody was labeled. To finally stain the nuclei of the cells, the samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The immunofluorescent images were visualized and captured using the PANNORAMIC MIDI (3DHISTECH™, Budapest, Hungary) at 200× magnification. Semi-quantitative image analysis was performed with ImageJ software. The data were presented as relative fluorescence intensity to the Con group.

Paraffin-embedded tissue sections were deparaffinized in 2 changes of xylene and rehydrated in serial decreasing concentrations of ethanol (100%, 85% and 75%). The sections were then immersed in EDTA antigen retrieval buffer (pH 8.0) (Servicebio, G1206) and held at sub-boiling point temperature for 8 min twice. After washing three times with PBS (pH 7.4) in a Rocker device (Servicebio, TSY-B), the sections were further covered to block non-specific binding in 3% BSA for 30 min, followed by incubation overnight at 4°C in a wet box with primary antibody CD11b or IL-6 prepared in PBS at a certain ratio (1:500). After washing 3 times for 5 min each on a rocker in PBS (pH 7.4), sections were covered with fluorescent-CY3 secondary antibody (Servicebio, GB21303), and incubated for 50 min at room temperature in dark. After incubating with DAPI solution (Servicebio, G1012) at room temperature for 10 min in dark, sections were incubated with spontaneous fluorescence quenching reagent (Servicebio, G1221) for 5 min. Then, the sections were mounted with an anti-fade mounting medium (Servicebio, G1401). Microscopy detection and collection of images were performed by Fluorescent Microscopy (Nikon, NIKON ECLIPSE C1). The color channel of the fluorescence was adjusted using CaseViewer software. The quantification was determined by ImageJ software.

Paraffin sections of the femur were routinely dewaxed, rehydrated, and placed in EDTA antigen retrieval buffer (pH 8.0; Servicebio, Wuhan, China). Sections were cooled, rinsed with PBS, and incubated in 3% aqueous hydrogen peroxide (Servicebio, Wuhan, China) at room temperature in the dark for 25 minutes to block endogenous peroxidase activity. Sections were rinsed with PBS and blocked with 3% BSA (Servicebio, Wuhan, China) at room temperature for 30 minutes. Sections were incubated with a primary anti-TPO rabbit polyclonal antibody (1 : 200, Abcam, UK) overnight at 4°C. After primary antibody incubation, sections were rinsed with PBS, incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (1 : 200, KPL, USA) for 50 minutes at room temperature, visualized with diaminobenzidine, and counterstained with hematoxylin. Three random fields per slice were observed under a 200x microscope, and TPO expression was analyzed.