Fms-EGFP (MacGreen) mice were used in every experimental procedure. These transgenic mice had a C57BL/6 background and expressed the enhanced green fluorescent protein (EGFP) under the CSF1R promoter specific of the mononuclear phagocytic system. The mice were 4 weeks old and were always maintained 12:12 h light cycle housing conditions and free access to food and water. All procedures were approved by the European Directive 2010/63/EU and NIH guidelines, and local Ethics Committees.
Mice were perfused transcardially with 4% paraformaldehyde (PFA), and brains postfixed for 3 h at RT. Then, the brains were rinsed in PBS and stored at 4°C until processing. The mouse brains were coronally sliced in a vibratome (Leica vibrating blade microtome) in order to obtain 6 series of 50 μm thickness sections. Standard immunofluorescence procedures were followed for tissue immunostaining. The staining was performed in free-floating tissue slices which were first blocked and permeabilized for 3 h at RT in 0.3% Triton-X100, 0.5% BSA in PBS solution and second, sections were treated for 2 overnight with a combination of the primary antibodies rat-anti-C1q and chicken-anti-GFP diluted in 0.3% Triton-X100, 0.5% BSA in PBS at 4°C. After this period of time, the tissue sections were rinsed with 0.3% Triton-X100 in PBS and incubated with the secondary antibodies (RRX and Alexa-488) and DAPI in 0.3% Triton-X100, 0.5% BSA in PBS at RT. Afterward, brain sections were thoroughly rinsed with PBS and mounted on glass slides with mounting medium (DakoCytomation).
Confocal microscopy/Image Analysis
Images of the DG of the immunostained brain sections were obtained using confocal microscopy (Leica SP8) under a 40X objective and scanning at a Z-step of 0.7 μm. A total of 3–4 sections were fully analyzed under the microscope with a zoom of 2.5 in order to find apoptotic cells.
The presence of pyknotic and/or karyorrhectic nuclei observed by DAPI staining was used to define apoptotic cells. On the other hand, characteristic of phagocytosed apoptotic cells were only attributed to those dead cells completely surrounded by an enclosed three-dimensional pouch of a microglial branch.
FACS Sorting was carried out following the previously well-described protocols of Abiega et al. and Sierra et al.. Briefly, both enzymatic (papain, 20 U/ml and DNAse I, 150 U/μl) and mechanic homogenization were used in order to digest the hippocampal tissue of the mice. Afterward, the homogenate was filtered through a 40 μm nylon strainer and the enzymatic activity was quenched with 5 ml of 20% FBS in HBSS. Then, cells were centrifuged at 200 g for 5 min and resuspended in 20% SIP (Solution of Isotonic Percoll, in HBSS). Afterward, fire-polished pipettes were used to slowly add HBSS on top of the 20% SIP in order to create a Percoll gradient. Next, samples were centrifuged at 200 g for 20 min with the lowest acceleration or deceleration in order to avoid any disruption of the gradient. Then myelin (the interphase) was removed and cells were rinsed with HBSS in a 200 g centrifugation for 5 min. Finally, the resulting pellet was resuspended in sorting buffer and cells were sorted in a FACS Jazz (BD) in order to collect the green fluorescent cell population. This population was directly collected in Lysis Buffer (Qiagen) with RNAse inhibitors (0.7% β-mercaptoethanol) and stored at -80ºC.
RNA Isolation and RTqPCR
For the RNA isolation of microglia, the RNeasy Plus micro kit (Qiagen) manufacturer’s instructions were followed. iScript kit (iScript Advanced cDNA Synthesis Kit, Biorad) was used in order to retrotranscribed the resulting RNA. For the RT-qPCR, cDNA was amplified with SsoFast EvaGreen Supermix (Biorad) and detected in a CFX96 System (Biorad). All the RT-qPCR experiments were performed according to the MIQE guidelines (Minimal Information for Publication of Quantitative Real-Time Experiments.
In order to avoid unspecific detection of genomic DNA contamination, primers were specifically designed (PrimerBlast, NIH) against exon-exon junctions of the genes of interest and the resulting amplicon was confirmed by melting curves in the RT-qPCR and by running in a 2% agarose gel electrophoresis. For each set of primers, the amplification efficiency was calculated using the software LinRegPCR and the relative quantification was measured with the following formula:
ΔΔCt = (1 + eff.target gene)exp(Ct sample − Ct control)/(1 + eff.reference gene)exp(Ct sample − Ct control)
C1q genes RT-qPCR was always performed in parallel with two independent reference genes, OAZ-1 and HPRT. OAZ-1 encodes an ornithine decarboxylase antienzyme and HPRT encodes hypoxanthine guanine phosphoribosyl transferase. Both reference genes expression was constant and therefore, OAZ-1 was selected as a reference in the statistical analysis due to the lower intragroup variability. Primer sequences are listed below:
OAZ1 (NM_008753; 51bp): Fwd AGCGAGAGTTCTAGGGTTGCC, Rev CCCCGGACCCAGGTTACTAC
HPRT (NM_013556.2; 150bp): Fwd ACAGGCCAGACTTTGTTGGA, Rev ACTTGCGCTCATCTTAGGCT
C1qA (NM_007572.2; 96bp): Fwd CACGGAGGCAGGGACAC, Rev GGCAGACATCTTCAGCCACT.
C1qB (NM_009777.2; 77bp): Fwd ATTCCATACACAGGAAGCCCC, Rev GCAGTAACAGGTGTGTCCAGA.
C1qC (NM_007574.2; 147bp): Fwd GCTGCTGCTGTTTCTTCTGG, Rev GGGATTCCTGGCTCTCCCT
Gene expression was compared between microglia and non-microglia cells using Student's t-test after assessing for normality and homoscedasticity with Sigmaplot.