Mouse and rabbit specific hrp dab detection kit

The IHC staining assay was performed following a standard procedure. Briefly, tissues from OA patients and healthy controls were fixed in 10% PBS-buffered formalin for 48 h. The resulting tissues were then performed a series of procedures including dehydration, infiltration and embedding in paraffin, section, and re-dehydration. The obtained slides were probed with primary antibodies including anti-CtBP1, anti-CtBP2, anti-CD31, anti-CD55, and anti-CD68, respectively, followed by incubation with secondary biotinylated antibodies for 1 h at room temperature. The mouse and rabbit specific HRP/DAB detection kit (Abcam, #ab64264) was used to determine the signals of each protein. For IF assay, the HOB-OA cells were fixed with 4% formaldehyde in PBS at room temperature for 20 min. After rinsing five times with PBS buffer, cells were incubated with the primary antibodies including anti-CtBP1, anti-p300 and anti-c-Jun, respectively, followed by incubation with Alexa Fluor 594 (red) (ThermoFisher Scientific, #A32740) and 488 (green) (ThermoFisher Scientific, #A32723) conjugated secondary antibodies at room temperature for 1 h. Cells were also stained with the fluorescent 4',6-diamidino-2-phenylindole (DAPI) to visualize the nucleus.

Kidney tissue sections were deparaffinized by immersing them in a xylene solution. Then the sections were rehydrated by gradually decreasing the ethanol concentration. Next, the sections were heated for 10 min at 95–100 °C in 10 mM sodium citrate buffer, pH 6.0, before being incubated for 2 h in 5% bovine serum albumin in tris-buffered saline. For the chromogenic staining stage, the slides were stained with a Mouse and Rabbit Specific HRP/DAB Detection Kit (Abcam, Cambridge, UK). Then, slides were incubated with primary antibodies against IL-6 (Cat. No. ab9324, Abcam, Cambridge, UK), TNF-α (Cat. No. ab205587, Abcam, Cambridge, UK), COX-2 (Cat. No. ab179800), NF-κB (p65) (Cat. No. ab194726, Abcam, Cambridge, UK) and KIM-1 (Cat. No. MBS175125, San Diego, CA, USA). Image analysis was performed on three sections of tissue from each rat using the Image J analysis software (NIH, Bethesda, MD, USA).