Sp 9001

Three cases of KIRC tissues and adjacent nontumor tissues from the First Affiliated Hospital of Guangxi Medical University were collected. After fixation with 4% paraformaldehyde for 24 h, paraffin embedding and section were performed. The sections were dewaxed and hydrated using xylene and gradient alcohol. Then, the sections were treated with EDTA at pH 8.5 (C1034, Solarbio) to induce epitope retrieval by heating. After washing three times with PBS, the sections were incubated with an endogenous peroxidase inhibitor (SP-9001, ZSGB-BIO) for 10 min. Then the sections were washed with PBS three times and incubated with normal goat serum blocking solution (SP-9001, ZSGB-BIO) for 15 min. Primary antibodies (TLR3, 1:200; TLR4, 1:200) were incubated overnight at 4°C. After 30 min of room temperature balance the next day, incubated with Biotinylated Second Antibody (SP-9001, ZSGB-BIO) for 15 min. Then the sections were washed with PBS three times and incubated with Streptavidin-Enzyme Conjugate (SP-9001, ZSGB-BIO) for 15 min. After washing three times with PBS, the sections were incubated with DAB chromogenic fluid (ZLI-9018, ZSGB-BIO) for 5 min. Finally, after redyeing with hematoxylin, the slices were fixed with neutral gum. The images were captured using microscope (Olympus, CX23) and then processed with ImageJ software (NIH).

The slides were retrieved using the heat-induced epitope retrieval method and then blocked in 5% goat serum (#SP-9001, ZSGB-bio, Beijing, China) for 10 min. Next, the primary antibody was added and incubated with gentle agitation overnight at 4 °C. After washing with PBS three times, the secondary antibody working solution was subsequently added and incubated for 10 min at room temperature. The slides were washed with PBS three times, and then the streptavidin/HRP working solution (#SP-9001, ZSGB-bio, Beijing, China) was added and incubated for 10 min at room temperature. After washing with PBS three times, fresh DAB working solution (#SP-9001, ZSGB-bio, Beijing, China) was added and incubated for 5 min at room temperature. The slides were washed, hematoxylin-stained, and finally observed under inverted bright field microscopy.

The brain was harvested following standard perfusion and then fixed with 4% paraformaldehyde for a span of 8–10 h. Subsequently, the brain tissue was dehydrated utilizing 30% ethanol for a full 24-h period. Then the brain tissue was conventionally embedded using a paraffin-embedding machine. Post-embedding, the paraffin-infused tissue was sectioned into 1 μm thicknesses using a slicer. A heat-induced epitope retrieval approach was employed to retrieve the slides, which were subsequently blocked with 5% goat serum (#SP-9001, ZSGB-bio, Beijing, China) for 10 min. The primary antibody was then added, and the mixture was gently shaken and incubated at 4 °C for an entire night. The secondary antibody working solution was then added and incubated for 10 min at room temperature following three PBS washes. Streptavidin/HRP working solution (#SP-9001, ZSGB-bio, Beijing, China) was added to the slides after three PBS washes, then left to incubate for 10 min at room temperature. Fresh DAB working solution (#SP-9001, ZSGB-bio, Beijing, China) was added and incubated for 5 min at room temperature after three PBS washes. The slides were cleaned, stained with hematoxylin, and then examined using inverted fluorescence microscopy (Olympus IX73).