Living embryos were collected and dechorinated before being mounted onto a permeable membrane in halocarbon 27 oil and placed under a glass coverslip as in Garcia et al., (2013) (link). Individual embryos were then imaged as described in Waymack et al., on a Nikon A1R point scanning confocal microscope using a 60X/1.4 N.A. oil immersion objective and laser settings of 40uW for 488nm and 35uW for 561nm (Waymack et al., 2020 (link)). To track transcription, 21 slice Z-stacks, at 0.5um steps, were taken throughout the length of nc14 at roughly 30s intervals. To identify the imaged position in the embryo, the whole embryo was imaged after nc14 prior to gastrulation at 20X using the same laser power settings. This whole embryo image was used to assign each transcription spot into one of 42 bins across the anterior-posterior (AP) axis of the embryo. The first bin corresponds to the anterior end of the embryo.
A1r point scanning confocal microscope
Manufactured by Nikon
Sourced in Canada
The Nikon A1R point scanning confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a point scanning design that enables high-resolution, optical sectioning capabilities for detailed analysis of biological samples. The A1R provides precise control over the excitation light, resulting in improved signal-to-noise ratios and enhanced image quality.
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Lab products found in correlation
Cm3050s cryostat, by Leica (4 mentions)
Tissue tek oct compound, by Sakura Finetek (4 mentions)
Ab1542, by Merck Group (4 mentions)
Ab52623, by Abcam (4 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (4 mentions)
Nis elements software, by Nikon (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
A 21245, by Thermo Fisher Scientific (2 mentions)
A 11015, by Thermo Fisher Scientific (2 mentions)
A21099, by Thermo Fisher Scientific (2 mentions)
A21448, by Thermo Fisher Scientific (2 mentions)
Nis elements 4, by Nikon (2 mentions)
Plan apo vc 60 na 1.4 oil objective, by Nikon (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Vimentin m7020, by Agilent Technologies (1 mentions)
Ki 67 antibody, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Zen microscope software, by Zeiss (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Hoechst, by Merck Group (1 mentions)
22 protocols using a1r point scanning confocal microscope
1
Embryonic Transcription Imaging Protocol
2
Immunohistochemical Staining of Mouse Tissue
3
Live Embryo Imaging for Transcriptional Dynamics
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Live embryos were collected prior to nc14, dechorionated, mounted on a permeable membrane, immersed in Halocarbon 27 oil, and put under a glass coverslip as in Garcia et al., 2013 (link). Individual embryos were then imaged on a Nikon A1R point scanning confocal microscope using a 60X/1.4 N.A. oil immersion objective and laser settings of 40uW for 488 nm and 35uW for 561 nm. To track transcription, 21 slice Z-stacks, at 0.5 um steps, were taken throughout the length of nc14 at roughly 30 s intervals. To identify the Z-stack’s position in the embryo, the whole embryo was imaged after the end of nc14 at 20x using the same laser power settings. Later in the analysis, each transcriptional spot’s location is described as falling into one of 42 anterior-posterior (AP) bins, with the first bin at the anterior of the embryo. Unless otherwise indicated, embryos were imaged at ambient temperature, which was on average 26.5°C. To image at other temperatures, embryos were either heated or cooled using the Bioscience Tools (Highland, CA) heating-cooling stage and accompanying water-cooling unit.
4
Somite-like Structure Quantification
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For the primary and secondary screens, images were acquired on a Nikon A1R point scanning confocal microscope. For each organoid, 66 z-series optical sections were collected with a step size of 2 µm. Quantification of somite-like structures for the secondary screen was done by blinded manual scoring, considering the following criteria:
5
Immunofluorescent Staining of Tissue Sections
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Tissue sections (5 μm thick) were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed with proteinase K (20 μg/mL in PBS) for 30 minutes at 37°C. Samples were blocked with 5% bovine serum albumin/PBS and incubated overnight at 4°C with primary antibodies diluted in 5% bovine serum albumin/PBS [IL-1R1, ab106278 (Abcam, Cambridge UK); IL-6, AF-206 at 10 μg/mL (R&D Systems, Minneapolis, MN); vimentin, M7020 at 62 μg/mL (Dako)]. Slides were washed with PBS-Tween (0.05%) and incubated with anti-rabbit tetrarhodamine isothiocyanate (T6778; Sigma-Aldrich) and anti-mouse fluorescein isothiocyanate (F2012; Sigma-Aldrich) diluted in 5% bovine serum albumin/PBS for 1 hour at room temperature. Slides were washed with PBS-Tween and mounted using mounting medium with DAPI (H1200; Vector Laboratories). Images were acquired on a Nikon A1R point scanning confocal microscope.
6
Matrigel-based 3D Cell Culture Protocol
7
Immunofluorescence Staining of FLAG-Tagged Proteins
8
Nile Red Emission Spectrum of B. subtilis
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The analysis of the emission wavelength spectrum for Nile Red-stained B. subtilis cells was carried out with Nikon A1R point scanning confocal microscope using Nikon Plan Apo VC × 60 NA 1.4 oil objective and 561 nm excitation laser. The emission spectrum was recorded with a 4.8-nm window size for the total range of 570–749 nm. Image analysis was carried out with NIS-Elements 4.0 (Nikon).
9
Live-Cell Imaging of Laminin Endocytosis
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MCF10A cells were transfected with LAMP1-mRFP-FLAGX2, starved for 24 h and then treated with fluorescently labelled laminin-Alexa-647. The cells were imaged with Nikon A1R point scanning confocal microscope, with Perfect Focus System, and a × 60 Plan Apo Oil lens, over a span of 60 min and images were acquired every 10 s. The cells were kept in a humidified, 5% CO2-supplemented, 37 °C chamber during imaging.
10
Immunohistochemistry of Murine Tissue
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Tissue was harvested immediately after euthanizing mice and fixed with 4% paraformaldehyde overnight. Tissue was then washed with PBS for 5 times, 10 min each and incubated in PBS/30% sucrose for 8 h and then frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek, 4583). Frozen tissue was cut into 30 μm sections on a Leica CM3050 S cryostat. Sections were briefly rinsed with PBS and blocked with PBS/0.3% Triton X-100/5% FBS overnight. Sections were then stained with Th antibody (AB1542, EMD Millipore, 1:200) and Tubb3 antibody (ab52623, Abcam, 1:200) for 2 days. Sections were washed with PBS/0.03% Triton X-100/5% FBS for 5 times, 1 h each and then stained with anti-sheep Alexa Fluor 488 (Thermo Fisher, A-11015, 1:500) and anti-rabbit Alexa Fluor 647 (Thermo Fisher, A-21245, 1:500) for 2 days. Sections were washed with PBS/0.3% Triton X-100/5% FBS for 5 times, 1 h each and then mounted in ProLong Diamond Antifade Mountant (Thermo Fisher, P36965). Images were taken on a Nikon A1R point scanning confocal microscope.
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