Mice were anaesthetized and transcardially perfused at either 3, 8 or 12 months of age with PBS. Brains were removed and the hemispheres separated. One hemisphere of the brain was immersion‐fixed in 4% paraformaldehyde for immunohistochemical analysis as previously described [22 (link), 23 (link)], and the other hemisphere was sub‐dissected and snap‐frozen in liquid nitrogen for biochemical analysis. Fixed brains were processed using an automated system (Excelsior, Thermo, USA), embedded in paraffin and sagittally sectioned at the level of the mid‐hippocampus into 3‐μm‐thick sections using a microtome (Thermo, USA). All staining was done in Sequenza staining racks for standardisation using previously reported protocols [24 (link)]. The following primary antibodies were used: pan‐TDP‐43 (10782‐2‐AP, Proteintech), POU3F2 (ab94977, Abcam), GFAP (G9269, Sigma), EGFP (ab184601, Abcam), Cyclin F (sc‐515207, Santa Cruz) and VCP (sc‐20799, Santa Cruz). Microscopy was performed with an Olympus BX51 (United States) epi‐fluorescence microscope equipped with an XM10 MONO camera, or sections were scanned with an Axio Scan Z1 automated slide scanner (Zeiss).
Free full text: Click here
