Alexa fluor 405 goat anti rabbit igg h l

GF-1 cells were seeded in 6-well plates with 2.5 mL of medium (10⁵ cells/mL) for 20 h and then transfected with EYFP, EYFP-B2 and EYFP-B2Δ for 48 h. The cells were washed with cold PBS, fixed in 4 % formaldehyde for 30 min at room temperature, washed with PBS twice and then permeabilized with PBST buffer (0.1 % Triton X-100 in PBS) for 15 min at room temperature. After the cells were washed with PBS twice, they were blocked with 1 % BSA for 60 min at RT, and then incubated overnight at 4 °C with antibodies against Drp1 (1:50, Aviva Systems Biology, San Diego, CA, USA) overnight. After cells were washed with PBST buffer twice, they were incubated for 60 min with Alexa Fluor^® 405 Goat Anti-Rabbit IgG (H+L) (1:500, Invitrogen) in 1 % BSA and washed with PBST twice. Immunofluorescence was examined using an Olympus IX70 fluorescence microscope (Tokyo, Japan) at 488-nm excitation and 515-nm long-pass filter for detection EYFP-B2 protein (with green fluorescence); 330-nm excitation and 420-nm long-pass filter was used for detection of the blue florescence (with Drp1) in the mitochondria.

Fluorescence activated cell sorting (FACS) was performed to characterize neuronal and astrocytic NuTRAP cassette expression. Whole hippocampal tissue from Emx1-NuTRAP mice was isolated and single-cell suspensions were immunostained for cytometric analysis⁸⁹ . We used antibodies for THY-1 (OX7) AlexaFluor™ 647 (Santa Cruz Biotechnology, sc-53116 AF647, Lot: F2716; concentration: 1:50) and S100β (Abcam, ab41548, Lot:GR3326165-1; concentration: 1:200) with an AlexaFluor™ 405 goat anti-rabbit IgG (H + L) (Invitrogen, A31556, Lot: 2273716; concentration:1:800) secondary antibody. FACS analysis was performed using a BD FACSAria™ Fusion Flow Cytometer (BD Biosciences) at the University of California, Irvine Stem Cell Core. Samples and single-stain controls were analyzed using the FlowJo v10.8.1 software (BD Biosciences). Samples and controls were positively gated for live cells (SSC-A and FSC-A) and single cells (FSC-H and FSC-A), and negatively gated for autofluorescence (Comp-Alexa Fluor 647-A (Thy1) and Comp-BV421-A (S100β)). Fraction of GFP + neuronal cells was determined using quartile analysis of Thy-1 and Comp-GFP-A (GFP). Fraction of GFP + astrocytic cells was determined using quartile analysis of S100β and Comp-GFP-A (GFP).