Tsy b

Slides were deparaffinized by incubating in xylene for 15 min, for a total of two washes. Slides were then dehydrated with absolute ethanol for 5 min, twice. Next, they were dehydrated sequentially in 85% and 75% ethanol for 5 min each. The slides were washed with distilled water, and antigen retrieval was performed in a buffer containing EDTA (pH 8.0). The temperature was maintained at sub-boiling for 8 min, standing for 8 min, and another sub-boiling for 7 min. The samples were washed thrice with PBS (pH 7.4) for 5 min each in a Rocker device (TSY-B; Servicebio), and the excess fluid was removed. The sample was marked using a liquid-blocking pen. To prevent nonspecific binding, samples were incubated in 3% BSA for 30 min. Samples were then incubated with the following primary antibodies at 4 °C overnight: cyclooxygenase 2 (COX-2), phosphorylated epidermal growth factor receptor (p-EGFR), and doublecortin and CaM kinase-like-1 (DCAMKL-1) from Abcam. The slides were washed three times with PBS for 5 min each and then incubated with a secondary antibody in the dark at room temperature for 50 min. The slides were washed as described above and incubated with DAPI for nuclear staining at room temperature for 10 min in the dark.

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.