Worms were washed off NGM plates and immobilized in 5 mM sodium azide (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in M9 buffer on glass slides. Images were collected using an A1R multiphoton confocal microscope (Nikon Instruments, Shanghai, China). At least 20 worms per strain were examined.
A1r multiphoton confocal microscope
Manufactured by Nikon
Sourced in United States
The Nikon A1R multiphoton confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It combines the capabilities of a multiphoton excitation system and a confocal microscope, enabling high-resolution imaging of thick, scattered, and scattering samples. The A1R multiphoton confocal microscope can perform deep tissue imaging, live-cell imaging, and other specialized applications.
Automatically generated - may contain errors
Lab products found in correlation
L2630, by Merck Group (2 mentions)
Srp3332, by Merck Group (2 mentions)
I3390, by Merck Group (2 mentions)
Nis elements software, by Nikon (2 mentions)
Sodium azide, by Santa Cruz Biotechnology (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Interferon gamma, by Merck Group (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Fluoromount, by Thermo Fisher Scientific (1 mentions)
Stellaris eight confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole dapi mounting media, by Vector Laboratories (1 mentions)
Ihc antigen retrieval solution, by Thermo Fisher Scientific (1 mentions)
Safeclear, by Thermo Fisher Scientific (1 mentions)
Fluoroshield dapi, by Merck Group (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
A1r multiphoton microscope, by Nikon (1 mentions)
Dapi mounting media, by Vector Laboratories (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
13 protocols using a1r multiphoton confocal microscope
1
Immobilizing Nematodes for Microscopy
2
Immunostaining of Drosophila Neural Tissues
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Adult brains and CNS or wing discs from third instar larvae were dissected in chilled 1× PBS and fixed with 4% paraformaldehyde for 20 min. Samples were washed thrice with 1× PBST (1× PBS with 1% Triton X-100) for 15 min. Primary antibodies: Mouse anti-DLG (DSHB: 4F3) 1:50, Mouse anti-Bruchpilot (nc82, DSHB), Mouse anti-Repo (DSHB: 8D12), Mouse anti-Elav (DSHB: 9F8A9). Secondary antibodies: Anti-mouse-568 (Thermo Fisher Scientific A11031) 1:200, Anti-Armadillo (DSHB: N27A1) 1:200, Anti-Wingless (DSHB: 4D4) 1:100. The samples were incubated with respective primary antibody overnight at 4°C and washed thrice using 1× PBST for 15 min. The samples were then incubated with appropriate secondary antibody for 3 h at room temperature and washed thrice using 1× PBST for 15 min. For anti-nc82 staining, primary antibody was incubated for 24 h, and secondary antibody was incubated overnight at 4°C. Brains, CNS, or wing discs were mounted on a glass slide using Vectashield (Vectorlabs) with a coverslip. The samples were scanned using Nikon A1R Multiphoton Confocal Microscope (2 μm z-stacks).
3
Macrophage Polarization and Phagocytosis
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Macrophages were isolated from the bone marrow of 6‐week‐old SD rats as described in Ying et al.,20 plated on 8 well‐multichambered slides (Falcon 354108) at 100,000 cells per well in macrophage growth media consisting of IDMEM without HEPES (Sigma‐Aldrich I3390), with 10% heat‐inactivated foetal calf serum and 10 ng/mL colony‐stimulating factor (CSF) (Sigma‐Aldrich, # SRP3332) and fed every 2–3 days. After 7–10 days, growth media was replaced with M1 or M2 induction media containing either 100 ng/mL lipopolysaccharide (Sigma‐Aldrich, L2630) plus 50 ng/mL interferon‐gamma (Sigma‐Aldrich, I3257) or 10 ng/mL IL‐4 (Sigma‐Aldrich, I3650), respectively, without CSF, at normal pH or a pH of 6. Three days after transfer to M1 or M2 phenotype induction media, 1 μL of a central myelin enriched fraction was added to half of the wells to induce phagocytosis15 and DiR‐labelled hMSC‐sEVs were added to wells. After 24 h, cultures were washed three times with plain IDMEM, fixed with 4% paraformaldehyde and stained with antibodies directed against CD206 and iNOS, visualized with fluorescent 488 and 594 wavelength secondary antibodies, counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) mounting media, examined, and photographed with a Nikon A1R multiphoton confocal microscope with NIS Elements software as above (Table S2 ).
4
Macrophage Polarization and Phagocytosis Study
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Macrophages were isolated from bone marrow of 6 weeks old SD rats as described in Ying et al. (Ying et al., 2013 ), plated on 8 well multichambered slides (Falcon 354108) at 100,000 cells per well in macrophage growth media consisting of IDMEM without HEPES (Sigma‐Aldrich I3390), with 10% heat‐inactivated foetal calf serum and 10 ng/ml colony‐stimulating factor (CSF) (Sigma‐Aldrich, # SRP3332) and fed every 2–3 days. After 7–10 days, growth media was replaced with M1 or M2 induction media containing either 100 ng/ml LPS (Sigma‐Aldrich, L2630) plus 50 ng/ml interferon‐gamma (Sigma‐Aldrich, I3257) or 10 ng/ml interleukin 4 (Sigma‐Aldrich, I3650) respectively without CSF, at normal pH or a pH of 6. Two days after transfer to M1 or M2 phenotype induction media, 1 μl of a central myelin enriched fraction (Lankford et al., 2017 (link)) was added to half of the wells to induce phagocytosis. DiR‐labeled MSC‐sEVs were added to wells three days after transfer to induction media. After 24 h, cultures were washed 3 times with plain IDMEM, fixed with 4% paraformaldehyde and stained with antibodies directed against CD206 and iNOS, visualized with fluorescent 488 and 594 wavelength secondary antibodies, counterstained with DAPI mounting media, examined, and photographed with a Nikon A1R multiphoton confocal microscope with NIS Elements software as above (Supplementary Table.1 ).
5
Polyrotaxane Internalization and Trafficking
6
Measuring Synaptic Density in the Brain
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Synaptic density measurement was performed as described in [146 (link)]. Brains were stained with anti-nc82 antibodies and imaged at 100× oil immersion (Nikon A1R Multiphoton Confocal Microscope). The SEZ region and both SLP regions were scanned separately for each brain. Fiji 3D Object Counter plugin was used to measure the synaptic contacts through 39 stacks in the SEZ region and 41 stacks in the SLP region. Same threshold [146 (link)] was applied across all the samples to return the total number of contiguous voxel elements across the images. The number of objects detected was plotted.
7
Visualizing Quadruplex-Loaded Polymer Capsules
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A FEI Quanta FEG SEM microscope (Hillsboro, OR, USA) at 10 kV was used to image dried suspensions of G-quadruplex-loaded (PMAA/PVPON) capsules. Capsules were dialyzed against DI water to prepare them for SEM, and a drop of the diluted capsule solution was placed on a silicon wafer and allowed to dry overnight. A Denton sputter-coater was used to coat the dried capsule samples with a ~5 mm thick layer of silver immediately before imaging. For CLSM imaging, 30 µL of the 1.6 × 108 capsules/mL stock solution was pipetted into Lab-Tek confocal microscopy (EMS, Hatfield, PA, USA) chambered coverglass containing buffer solutions. A 10 µL aliquot of Alexa Flour 568 hydrazide (0.5 mg/mL) fluorescent dye solution was added to assist in visualizing the capsules and the capsules were allowed to settle overnight before imaging with a Nikon A1R multiphoton confocal microscope (Tokyo, Japan) equipped with a 60× oil immersion objective.
8
Imaging Drosophila Eye Development
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Larval, pupal, and adult eyes were processed as described (Charlton-Perkins et al., 2011 (link)) and stained with mCut (DHSB, 1:20), Elav (DHSB, 1:50), DCAD2 (E-cadherin; DHSB, 1:50), and drosocrystallin (kindly gifted by H. Matsumoto; rbCry 1:100; Figure 1 ). The samples were imaged using a Nikon A1R multiphoton confocal microscope, and image processing was performed using NIS-Elements (Nikon) and Photoshop CC (Adobe). For Cut and N-cadherin staining, the pupal eye discs were dissected at ∼ 37% development as outlined in Tea et al., 2014 (link). In brief, tissue was fixed in 4% formaldehyde diluted in dissection solution for 20 min at RT. Post fixation, the eye discs were washed three times in PBT (PBS with 0.3% Tween 20). Then, 10% normal goat serum (NGS) in PBT was used to block the eye discs at RT. The eye discs were incubated in primary antibodies (anti-Cut 1:50 (DSHB) and anti-N-cadherin 1:50 (DSHB) in PBT) at 4°C for ∼40 h. Anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 488 were used as secondary antibodies at 1:500 for 2 h at RT. Subsequently, the eye discs were washed in PBT, the nuclei counterstained with DAPI, and after additional washing, mounted in Fluoromount (Fisher Scientific). Z-stacks were acquired using a Leica Stellaris eight confocal microscope with a ×40 objective at a resolution of 1,024 × 1,024 pixels and a pixel size of 0.047 µm.
9
Immunohistochemical Profiling of Rat Brain
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Rats were deeply anesthetized with sodium pentobarbital, perfused with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, and processed for standard frozen sectioning. Coronal (20 μm) sections taken at 1.5 mm from the centre of each block were stained with two or more of the primary antibodies (Supplementary Table 1 ) diluted in 0.01% Triton X‐100, 5% fishskin gelatin (0.1 M PBS) blocking buffer, visualized with species‐specific secondary antibodies (Supplementary Table.1 ) counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) mounting media (Vectashield, Vector Laboratories, Burlingame, CA, USA) and photographed with a Nikon A1R multiphoton confocal microscope with NIS Elements software (Nikon, Tokyo, Japan).
10
Immunohistochemical Analysis of Enteroid Structure
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