3 3 diaminobenzidine dab chromogen

After rats were sacrificed on day 28, their prostate tissues were removed and sectioned in the sagittal plane. Then, prostate tissues would be embedded into paraffin blocks to support the tissue structure. The paraffin-embedded slides were de-paraffinized and rehydrated before antigen retrieval. Antigen was retrieved with citrate buffer in a high-pressure cooking pot, followed by quenching of endogenous peroxidase and blocking with normal serum. The slides were incubated overnight at 4 °C with antibodies to the proliferative cell nuclear antigen (PCNA; polyclonal, ab18197, dilution to 1:200; Abcam, Cambridge, UK). Tissue sections were subsequently washed and incubated with HRP-conjugated goat anti-rabbit secondary antibody (polyclonal; 111-035-144; dilution to 1:2000; Jackson ImmunoResearch, West Grove, Chester PA, USA) for overnight at 4 °C. Enzyme activity was then detected by adding 3,3′-diaminobenzidine (DAB) chromogen (LabVision Corp., Fremont, CA, USA). The images were captured by using a microscope (Leica, Wetzlar, Germany) with a 10× eyepiece and a 40× objective lens (400×). Then, 5 randomly distributed fields within the ventral prostate lobe on each slide were analyzed. The cells with brown nuclei were considered PCNA-positive, counted, and expressed as a percentage of the total cells.

On day 42, the mice were sacrificed by CO₂ inhalation and joint tissues were removed from the hind paws followed by fixing with 4% paraformaldehyde, decalcifying in 5% formic acid, and embedding in paraffin. 5 μm thick of paraffin sections were stained with hematoxylin and eosin (H&E) or safranin O/fast green (Sigma, St. Louis, MO, USA) for visualization of tissue structure or proteoglycan content prior to microscopic evaluation. Histopathological changes, such as hyperplasia, cell infiltration, and cartilage destruction in synovial tissues, were blindly assessed by a pathologist and assigned scores of 0–4 as the previous described: 0, no changes; 1, mild changes; 2, moderate changes; 3, severe changes; 4, total destruction of architecture [21 (link)]. The CD11c expressions in the synovium were examined with immunohistochemistry staining that started with incubation of the primary rabbit anti-CD11c antibody overnight at 4 °C (1:200; Servicebio, Wuhan, China). Tissue sections were subsequently washed and incubated with HRP-conjugated goat anti-rabbit secondary antibody for 30 min at room temperature. Enzyme activity was then detected by adding 3,3′-diaminobenzidine (DAB) chromogen (LabVision Corp., Fremont, CA, USA). The quantification of infiltrated CD11c⁺ cells was performed through the determination of the integrative optical density (IOD) with ImageJ software.