Monoclonal mouse anti human cd68 clone pg m1

Carotid plaque specimens were collected from patients who underwent carotid endarterectomy at Hadassah—Hebrew University Medical Center with or without a history of cerebrovascular symptoms (i.e., amaurosis fugax, transient ischemic attack, or stroke). The study protocol was approved by the Hadassah Helsinki Review Board (approval number HMO-09-0515) with written consent as described in [4 (link)]. The carotid endarterectomy specimens were collected from 3 patients. Freshly excised tissue samples were treated with 10μM GB111-NH₂ [6 (link)] (structure in S1 Fig) or vehicle (DMSO) for 24 hours in RPMI medium. Tissues were washed with PBS, serial frozen sections were stained with primary antibodies that were diluted in Cas-Block (Invitrogen) overnight at 4°C; monoclonal mouse anti-human CD68 clone PG-M1 (1:100, DAKO, Denmark), monoclonal rabbit anti human cleaved caspase 3 (1:400; Cell Signaling, CA, USA) and visualized with the Olympus confocal microscope. The percentage of apoptotic cells was determined by co-localization analysis using the JACoP/ImageJ program. At least two serial sections were analyzed per sample, the mean is presented ± standard Error (Statistical evaluations were done using GraphPad prism 7).

Formalin (4%)-fixed and paraffin-embedded human granulation tissue samples were sectioned at 2.5-μM thickness and mounted onto TOMO® Microscope Slides (Matsunami Glass, USA). Deparaffinization and rehydration were performed with xylol and ethanol dilution series. Antigen retrieval was performed in a water bath, by incubation in citrate buffer. The endogenous peroxidase was blocked with 3% H₂O₂ prior to antibody incubation. Primary antibody incubation was performed for 24h at 4°C using CD68 (Monoclonal Mouse, Anti-Human CD68, Clone PG-M1, Dako, Denmark) and perilipin (PLIN, Perilipin-1 (D1D8) XP® Rabbit, Cell Signaling Technology, Germany). Specimens were then treated with primary antibody enhancer, horseradish peroxidase polymer, and AEC substrate and then counterstained with hemotoxylin (Scharlau, Spain) and cover-slipped. Images were acquired with a Zeiss AXIO Imager Z2 microscope (Zeiss, Austria).