Fig. 4

HMC3 cell expression of microglial lineage markers. a–f Representative example of confocal images (1024 × 1024 pixels) acquired at × 20 magnification with a confocal laser scanning system (A1+, Nikon). HMC-3 cells immuno-labeled for IBA1 (green fluorescence, d, f) and DAPI stained (blue fluorescence, a, b) are shown. Merged images are shown in (e, f). Control experiments performed by omitting the primary antibody are shown in (a, c, e). No green fluorescence was present (c, e), indicating neither spontaneous fluorescence nor non-specificity of the secondary antibody. Scale bar 50 μM. g–h Total RNA was prepared from human microglial HMC3 cells 24 h after incubation in complete growth medium and retrotranscribed using random hexamers. Real time (Q)-PCR analysis for the mRNA levels of IBA1, CX3CR1, CCR2, P2RY12, TMEM119, and CSF1-R was carried out according to our standard protocols [109]. g, h panels show a representative gel image of PCR products obtained at the end of the analysis from two different RNA samples for each condition. In the last line is shown the actin amplification, as positive control