Mouse anti cd68

Five-micron sections of formalin-fixed paraffin-embedded adipose tissue were blocked in normal serum and incubated overnight with rabbit anti-CPT1A antibody (Sigma-Aldrich) at 1:50 dilution, and with mouse anti-CD68 (Santa Cruz Biotechnology, Inc.) at 1:50 dilution, washed, and visualized using Alexa Fluor 546 goat anti-rabbit, and Alexa Fluor 488 goat anti-mouse antibodies, respectively (1:500; Molecular Probes Inc.). The slides were counterstained with DAPI (4,6-diamidino-2-phenylindole) to reveal nuclei and were examined under a Nikon Eclipse 90i fluorescent microscope. As a negative control, the assay was performed in the absence of primary antibody.

To confirm microglial lineage and the effect of the different treatments on its activation, we assessed the expression of ionized calcium-binding adapter molecule 1 (IBA1) and CD68 by immunofluorescence analyses.
Microglial cells cultured for 1 day on permanox chamber slide at the concentration of 2.5 • 10 4 cell/cm 2 were treated with LPS alone or in combination with ADSC-CM. After 48 h at 37°C, the cells were fixed with 4% PFA (15 min, RT). To avoid quenching, microglia were treated with 0.1 M glycine (10 min, RT). Subsequently, the samples were permeabilized with 0.1% Triton X-100 for 15 min. The following primary antibodies were applied overnight at 4°C: mouse anti-IBA1 (1:500) and mouse anti-CD68 (1:400), (both purchased from Santa Cruz Biotechnology, Inc., Heidelberg, Germany). Cells were then incubated with Alexa488 conjugated secondary antibody (1:1,000; Life Technologies) at RT for 45 min, washed, and the nuclei were counterstained with DAPI (Life Technologies). The sample was mounted with ProLong Ò Gold Antifade Mountant (Life Technologies) and images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Cells were counted in three microscopic fields in each well (three wells/treatment) and expressed as a percentage of the total number of cells. Each treatment was performed in triplicate.