Ti e microscope

Lentivirus transduced, floating neural precursor aggregates were gently trypsinized to single cells and left to attach 1 h at 37 °C to poly-L-lysine (200 μg/ml) coated SuperFrost Plus microscope slides (Menzel-Glaser). Here they were fixed by 4% PFA for 20 min at 4 °C, washed three times in 1X PBS and processed for immunofluorescence. Fixed-cryopreserved brains were sliced at 16 μm, tissue slices were allowed to dry at least one hour at RT and processed for immunofluorescence.
In all cases, immunofluorescence was performed as previously described45 (link). The following primary antibodies were used: anti-Tubb3 (mouse clone Tuj1, Covance #MMS-435P, 1:1000); anti-GFP (chicken polyclonal, Abcam ab13970, 1:400); anti-Foxg1 (rabbit polyclonal, 1:20041 (link)). Secondary antibodies were conjugates of Alexa Fluor 488 and 594 (Invitrogen), used at 1:600. Cell nuclei were counterstained with DAPI (4′, 6′-diamidino-2-phenylindole).
Tubb3 immunofluorescences were photographed on a Nikon Eclipse TS100 fluorescence microscope equipped with a DS-2MBWC digital microscope camera with a 20X objective. Immunoprofiled brain sections were photographed on a Nikon TI-E microscope, equipped with 20X or 40X objectives and a Hamamatsu C4742–95 camera. All images were processed using Adobe 9.0.2 Photoshop 2 CS2 software and ImageJ.

d‐amino acids are involved in peptidoglycan biosynthesis of diverse bacteria (Lam et al., 2009). HADA is a fluorescent d‐amino acid analogue that can be used for monitoring the peptidoglycan synthesis activity of bacteria (Kuru et al., 2015). Cells were grown in LB broth until OD₆₀₀ reached 0.2. Relacidine B was added at a concentration of 0.25 μg ml⁻¹ and cells were kept growing for another 2, 4, and 5 h. DMSO was used as a negative control while ceftriaxone (4 μg ml⁻¹) was used as a positive control. At each time point, cells were treated with 500 μM HADA for 24 min. After treatment, cells were washed twice with PBS buffer. Incorporation of HADA was inspected with a Nikon Ti‐E microscope (Tokyo, Japan) equipped with a Hamamatsu Orca Flash 4.0 camera.

All microscopy, excluding the experiments for Figure 5, was performed at 30°C on a Delta Vision Deconvolution Microscope (Applied Precision), using InsightSSITM Solid State Illumination of 488 and 594 nm, an Olympus UPLS Apo 60x or 100x oil objective with 1.4NA and softWoRx software (GE lifesciences). Detection was done with a CoolSNAP HQ2 camera. Microscopy to study Msn2 dynamics was performed on a Nikon Ti-E microscope equipped with a Hamamatsu Orca Flash V2 using a 40X oil immersion objective (1.3NA). Fluorescence excitation was performed using an LED illumination system (Excelitas 110-LED) that is triggered by the camera.