Sab4600310

After overnight fixation in 4% PFA, the brains were processed for paraffin embedding, and once included, serial sagittal 4 μm thickness sections were cut using a microtome (Microm HM 340E). The slides were dewaxed using xylol and hydrated in decreasing concentration of ethanol until PBS 0.1 M, and after hydration, the immunofluorescence assay was performed as follows. Briefly, brain sections containing the hippocampus were subjected to heat-induced epitope retrieval and blocked with 10% BSA for 90 minutes at room temperature. Then, samples were immunolabeled at 4°C overnight using the following primary antibodies at 1 : 100 dilution: anti-Iba1 (GTX101495, Genetex), anti-CD68 (GTX37743, Genetex), and anti-TNFα (GTX110520, Genetex). The next day, the slides were washed and incubated with the secondary antibody (SAB4600310, Sigma Aldrich Co.) diluted at 1 : 500 during 3 hours at room temperature. Then, sections were washed repeatedly and covered with Vectashield medium (Vector Laboratories, Burlingame CA) containing DAPI to counterstain nuclei.

From the right hemispheres of the tested samples, sagittal sections with 4 µm thickness were obtained using a microtome (Microm HM 340E). To perform the immunofluorescence assay, these sections were subjected to heat-mediated antigenic retrieval and blocked with 10% BSA for 90 min at room temperature. They were then immunolabeled with the primary anti-MT-2A antibody (DF6755, Affinity Biotech) at a 1:100 dilution overnight at 4 °C. The next day, the sections were washed and incubated with the secondary antibody (SAB4600310, Sigma Aldrich Co.) at a 1:500 dilution for 3 h at room temperature and shaking. They were then washed several times and mounted with Vectashield medium (Vector Laboratories, Burlingame CA) containing DAPI to stain the nuclei. For imaging, a Zeiss confocal microscope was used at 20× magnification. Microphotographs were taken every 0.5 µm in the Z-plane maintaining constant pinole, contrast and brightness, and an orthogonal projection of each slice was obtained using Zen Blue software. Three sagittal slices of each individual separated by 150 µm were studied.