Pa5 20054

Prostatic inflammatory infiltrates were graded based on a histopathological classification system (Supplementary Table 1) using H and E staining.11 (link) After being baked, deparaffinized, and rehydrated, prostate tissue sections were stained in hematoxylin (Proteintech Group Inc., Wuhan, China) and eosin-phloxine solution (Servicebio Co., Ltd., Wuhan, China). Then, sections were dehydrated and mounted with neutral glue (Solarbio Co., Ltd., Beijing, China).
The protein expression intensities were identified using IHC staining. After being deparaffinized, rehydrated, antigen retrieved, and blocked, prostate tissue sections were stained with primary antibodies: TLR2 (1:400; NB100-56720, Novus Biologicals Inc., Littleton, CO, USA), TLR10 (1:500; PA5-20054, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA), high mobility group box 1 (HMGB1; 1:2000; ab18256, Abcam Inc., Cambridge, MA, USA), and biotinylated secondary antibody: AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA). Then, sections were dehydrated and mounted with neutral glue. The mean depth of the brown color (three levels) under the microscope (Olympus, Tokyo, Japan) in four random quadrants was regarded as the intensity of relevant protein expression.

Immunofluorescent (IF) staining was used to identify the sites of TLR2 and TLR10 expression in prostate tissues/cells. After being fixed, blocked, and permeabilizated (when needed), prostate tissue/cell sections were stained with primary antibodies: TLR2 (1:400 for tissue sections, NB100-56720, Novus Biologicals Inc.; 1:400 for cell sections, MA5-16200, Thermo Fisher Scientific Co., Ltd.) and TLR10 (1:500 for tissue sections and 1:1000 for cell sections; PA5-20054, Thermo Fisher Scientific Co., Ltd.). Next, sections were stained with fluorescent secondary antibodies: Alexa Fluor® 488 Donkey anti-Rabbit IgG (H + L) antibody (CA21206S, Thermo Fisher Scientific Co., Ltd.), Alexa Fluor® 594 Donkey anti-Mouse IgG (H + L) antibody (CA21203S, Thermo Fisher Scientific Co., Ltd.), and 4′6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Co., Ltd.). Then, sections were mounted with antifade mounting medium (Abcam Inc.).