Following recording, slices containing neurobiotin-filled neurons were fixed overnight in Lana’s fix. Slices were subsequently washed four times in 0.01 M PBS, then placed in 0.5 μl streptavidin Cy5 (Thermo Scientific, USA) in 500 μl 0.1 M PB containing 0.3% Triton X-100 overnight. For TH-staining, slices were incubated in a 1:1000 Anti-Tyrosine Hydroxilase antibody (AB152, lot 3114503, Millipore Sigma, USA). Slices were washed again before mounting on Superfrost Plus microscope slides (Thermo Scientific, USA) using Fluoromount Aqueous mounting medium (Sigma, USA). Mounted tissue images were subsequently acquired using the Zen 2 black edition software (Carl Zeiss AG, Germany).
Zen 2 black edition software
Manufactured by Zeiss
Sourced in Germany
The ZEISS Zen 2 black edition software is a comprehensive microscope control and image processing platform. It provides a user-friendly interface for controlling ZEISS microscopes and cameras, as well as advanced tools for image acquisition, analysis, and processing.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Streptavidin cy5, by Thermo Fisher Scientific (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Lsm 880 laser scanning microscope, by Zeiss (1 mentions)
Hmga1, by Abcam (1 mentions)
Fibrillarin, by Thermo Fisher Scientific (1 mentions)
Lsm 780 microscope, by Zeiss (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
5 protocols using zen 2 black edition software
1
Visualizing Neurobiotin-Filled Neurons
2
Measurement of FIT Homo- and Heterodimerization
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Förster resonance energy transfer–acceptor photobleaching (FRET–APB) experiments were conducted 48 h after tobacco leaf infiltration with agrobacteria containing pABindGFP, pABindmCherry, and pABindFRET vectors recombinant for FIT, FITm forms, and BHLH039 to measure the homo‐ (FIT‐GFP + FIT‐mCherry or FITm‐GFP + FITm‐mCherry) or the heterodimerization (bHLH039‐GFP + FIT‐mCherry or bHLH039‐GFP and FITm‐mCherry) efficiency (Table S1 ). Donor proteins were additionally transformed single‐tagged with GFP and double‐tagged with GFP‐mCherry to serve as a negative and positive control. Gene expression was induced by spraying the leaves with a 20 μM β‐estradiol solution 16 h before FRET–APB measurements.
Measurements were operated withzen 2 black edition software (Zeiss) at the confocal laser‐scanning‐microscope LSM780 (Zeiss). Fluorescence intensity for both fluorophores was detected within 20 frames in a 128 × 128 pixel format and a pixel time of 2.55 μs. After the fifth frame, 100% laser power (561 nm) was used to bleach mCherry using 80 iterations. FRET efficiency was calculated in percent as relative increase of GFP intensity after acceptor photobleaching. At least two independent experiments with 10 measured nuclei each were performed (n = 10 nuclei).
Measurements were operated with
3
Zebrafish Confocal Imaging Protocol
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Zebrafish confocal images were captured at RT using a Zeiss LSM 800 confocal microscope. Z-stack images were acquired using the 63×, NA 1.4 oil objective at a 2× zoom, and with sectioning set automatically to optimal. Images were acquired and processed with ZEN 2 black edition software (Carl Zeiss). Z-stack images are presented as flat Z-projections. Only linear adjustments were made to brightness and contrast, and the final figures were assembled using Photoshop and Illustrator software (Adobe).
4
Confocal Laser Microscopy Imaging of Microplastics
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The samples for confocal laser microscopic imaging were prepared in the same manner as described in Section 2.3 . Confocal laser microscopic imaging investigation was carried out using an LSM 880 laser scanning microscope (Carl Zeiss) equipped with a 40×/0.80 numerical aperture Achroplan W water immersion objective (Carl Zeiss) and ZEN 2 Black edition software (Carl Zeiss). The samples for this imaging were prepared in the same manner as the previous section. All the images of the microplastics were observed using a 488-nm excitation wave and emission wave of >505 nm.
5
Immunofluorescence and DNA FISH Analysis
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Cells were grown on glass coverslips, washed with PBS, fixed at room temperature with 2% PFA for 10 min, and permeabilized with 0.5% Triton X-100 for 5 min followed by PBS washes. Immunofluorescence was performed with antibodies against H3K9me3 (Jenuwein Laboratory, #1926, 1:1000 dilution in 10% FBS/PBS), Lamin B (Santa Cruz, sc-6217, 1:200 dilution), HP1α (Millipore, 05-689, 1:500 dilution), Fibrillarin (Thermo Fisher, MA3-16771, 1:500 dilution), and Hmga1 (Abcam, ab129153, 1:500 dilution) for 1 h at room temperature. For DNA FISH, cells were dehydrated in ethanol. Denaturation at 80 °C for 5 min and hybridization at 37 °C for 1 h were performed in hybridization solution (2× SSC pH 7, 50 mM sodium phosphate pH 7, 60% formamide) with a mix of 4 LNA probes (3 nM each). Probe sequences are listed in Supplementary Table 1 . Image acquisition was performed on an LSM 780 microscope (Zeiss) using ZEN2 black edition software (Zeiss).
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