Immunohistochemistry of DRs

Histological analysis of dopaminergic receptors (DRs) was performed on paraffin embedded tissue samples. After de-paraffinization and rehydration, endogenous peroxidases were blocked by incubation with 3% H₂O₂, followed by antigen retrieval. This was achieved by incubation at 92 °C in citrate buffer or by enzymatic digestion with proteinase K, depending on the receptor subtype. Non-specific binding sites were blocked with phosphate-buffered saline (PBS) containing 10% fetal calf serum, 10% chicken serum and 10% albumin. Samples were then incubated overnight at 4 °C with the primary antibody (mouse anti-hD1DR: Santa Cruz, cat. nr. sc-3360; rabbit anti-hD2DR: Acris, cat. nr. TA316680; mouse anti-hD3DR: BioLegend, cat. nr. 827501; rabbit anti-hD4DR: OriGene, cat. Nr. TA321201; mouse anti-hD5DR: Santa Cruz, cat. nr. sc-376088) and then counterstained by using Histofine (Nichirei Biosciences, cat. Nr. 414152F), as indicated by the manufacturer. Control staining with the respective isotype controls (D1DR: santa cruz, cat. nr. sc3879; D2DR and D4DR: DAKO, cat. nr. DAK-X090302-8; D3DR: Biolgend, cat. nr. 400201; D5DR: santa cruz, cat. nr. sc-3877) and with the secondary antibody alone (Histofine, Nichirei Biosciences, cat. Nr. 414152F) were carried out in parallel and showed no positive staining.

Dopamine receptor expression was quantified in the invasion zone and synovial tissue of OA and RA patients with ImageJ software. Invasion zone was defined as the area comprised between the cartilage or bone surface (x = 0) and 20 μm inside the synovial tissue. Four different measurements were evaluated per region and patient, and three to four patients were evaluated per group. Dopamine receptor expression intensity was represented as a function of distance in the invasion zone. Additionally, mean intensity value for each dopamine receptor was compared between both regions.