Lsm 880 airy inverted microscope

Cerebral organoids were fixed with 1% PFA in 120 mM phosphate buffer pH 7.4 for 20 min at room temperature and subjected to cryosectioning (14 µm) and immunofluorescence as described (Camp et al., 2015 (link)). The following primary antibodies were used: rabbit anti-PAX6 (PRB-278P; Covance), sheep anti-TBR2 (AF6166; R+D systems), rat anti-CTIP2 (ab18465; Abcam), rabbit anti-KI67 (ab15580; Abcam). The secondary antibodies, used in combination with DAPI staining, were all donkey-derived and conjugated with Alexa 488, 555 or 647 (Life Technologies). Images were acquired with a Zeiss LSM 880 Airy inverted microscope, using 10X (0.45 NA) and 20X (0.8 NA) Plan-Apochromat objectives, and analysed using Fiji. Quantifications were carried out in cortical regions of D28 and D52-54 cerebral organoids by counting, from the ventricular to the pial surface, either all PAX6 and TBR2 positive and negative nuclei stained by DAPI in 50 μm and 100 μm wide fields, respectively, or all KI67-positive cells in 100 μm wide fields. An average of 350 cells per sample were counted. Statistical significance was calculated using the Mann–Whitney U-test.

Water-in-water proteinosomes were imaged using a 10× objective (Plan-Neofluar 10× NA 0.3 Ph1, Zeiss) mounted onto a Zeiss Axiovert 200 M inverted widefield microscope equipped with a 16-channel CooLED pE-4000 and an Andor Zyla PLUS sCMOS camera in phase contrast mode or fluorescence. The microscope was equipped with the following filter sets: λ_ex = 542/27 nm and λ_em = 593/46 nm Beamsplitter H 560 LPXR for imaging DY530, λ_ex = 575/15 nm and λ_em = 641/75 nm Beamsplitter HC BS 596 for imaging carboxy-X-rhodamine (ROX), λ_ex = 475/28 nm and λ_em = 525/15 nm Beamsplitter H 488 LPXR for imaging EvaGreen.
Fluorescent confocal images were undertaken with objective Zeiss C-Apochromat 40× NA 1.2 W objective mounted on a confocal Zeiss LSM 880 Airy inverted microscope. Samples were loaded into capillary channels (ID 1.0 × 0.1 mm, 0.7 mm thickness) and then sealed with 5 min epoxy glue. DY530 labelled DNA was imaged with λ_exc = 514 and λ_em = 544–695 nm or for FITC labelled protein conjugate with λ_exc = 488 and λ_emis = 499–535 nm. Size analysis and distribution of DNA within individual proteinosomes containing fluorescently labelled DNA were analysed using a custom-written analysis macro script using FIJI (see SI and data availability statement).