Lsm 780 upright confocal microscope

Neuronal cell death was assayed by TUNEL staining (TdT FragEL Kit; Calbiochem, San Diego, CA, USA) in 4 μm thick sections prepared from formalin‐fixed, paraffin‐embedded Drosophila heads. Colorimetric detection of TUNEL labeled nuclei was performed using the VECTASTAIN Elite ABC Kit and DAB substrate (Vector Laboratories, Burlingame, CA, USA). TUNEL labeled nuclei were counted throughout the entire brain. For Lamin/TUNEL co‐staining, Drosophila brains were dissected in PBS and fixed for 20 min in methanol prior to staining. Primary antibodies were anti‐Lamin Dm0 (DSHB, ADL67.10) and anti‐DIG (R&D Systems, MAP7520). Secondary detection was performed using Alexa Fluor^TM‐conjugated secondary antibodies and Alexa Fluor^TM‐conjugated streptavidin (ThermoFisher Scientific) diluted in PBSTr + 2% milk. All images were taken with a Zeiss LSM 780 upright confocal microscope and analyzed with ImageJ software. All images shown are a single slice.

N. benthamiana leaf sections (4 mm²) away from the infiltrated point were excised, infiltrated with water and imaged on a Zeiss LSM 780 upright confocal microscope, LSM 710 inverted confocal microscope or LSM 880 inverted confocal microscope fitted with 40X C-Apochromat water immersion objective (NA = 1.2) (Carl Zeiss Inc, Thornwood, NY). The 405 nm, 458 nm, 488 nm, 514 nm, or 561 nm laser line was used for TagBFP, Cerulean, GFP, Citrine, or TagRFP, respectively. TagBFP and Cerulean were pseudo-colored cyan, Lifeact-TagRFP and mTalin-Citrine were pseudo-colored magenta, and GFP-TUA6, EB1-Citrine, and TagRFP-MAP-CKL6 were pseudo-colored yellow throughout the manuscript. In the perinuclear clustering experiment, Citrine for cytosol and nucleus diffusion was pseudo-colored blue and mCherry with nuclear localization signal was pseudo-colored blue for consistency of data presentation.

Specimens were fixed in 4% paraformaldehyde/1X PBS at 4 °C for 24 h (TL/TLE cases), and up to 72 h in the case of germinal matrix, rinsed in 1X PBS, and vibratome sectioned (30 µm). Sections were incubated for 1 h in blocking solution (1X PBS/0.5% Triton X-100/10% normal donkey serum); then for 24 h at 4 °C in primary antibody (1X PBS/0.25% Triton X-100/1% normal donkey serum); and then for 4 h at room temperature in either donkey anti-mouse, donkey anti-rabbit, donkey anti-rat, or donkey anti-goat fluorochrome-conjugated secondary antibodies (Jackson Laboratories, 1:250 dilution). Formalin-fixed paraffin embedded (FFPE) tissues underwent 1 h deparaffinization, rehydration in decreasing gradient of ethanol, and antigen retrieval for 20 min prior to blocking. Primary antibodies dilutions were as follows: 1:50 mouse anti-EGFR (Invitrogen 280005); 1:500 rat anti-GFAP (Life Technologies, 13–0300); 1:250 rabbit anti-OLIG2 (Millipore, AB9610); 1:250 goat anti-hOLIG2 (R&D Systems, AF2418SP); 1:250 rabbit anti-Ki67 (Abcam, Ab15580); 1:250 mouse anti-Ki67 (BD Biosciences, 556003); 1:100 rabbit anti-PAX6 (Novus Biologicals, NBP1-89100); 1:100 mouse anti- NEUN (Millipore, MAB377); 1:250 rabbit anti-AIF1 (IBA1) (Wako, 019–19741). Nuclei were counterstained with DAPI (1:1000). Images were obtained using a LSM 780 upright confocal microscope (Zeiss).