For confocal microscopy, macrophages were seeded in black poly-d-lysine coated glass 96-well plates (MatTek Corporation, Ashland, MA, USA). To stain lysosomes, macrophages were incubated with 75 nM Lysotracker Red or Deep Red (Thermo Fisher Scientific) at 37°C/5%CO2 for 1 h before fixation. Cells were fixed for 1 h in 1% EM-grade formaldehyde, followed by quenching with PBS/1.5 mg/ml glycine for 10 min and blocking in 5% human serum for 45 min, all at room temperature. For immunostaining, cells were permeabilized for 10 minutes with 0.1% Triton X-100 before blocking and subsequently stained with primary and secondary antibodies for 30 minutes each in the dark at room temperature. Finally, cells were stained with phalloidin-Alexa488 (Thermo Fisher Scientific) and/or LipidTOX Green (Thermo Fisher Scientific) for 30 min according to the manufacturers’ instructions, and/or 50 μg/ml Filipin complex from Streptomyces filipinensis (Sigma-Aldrich) for 2 h at room temperature in the dark. Lysotracker and filipin pictures were taken using a SP8WLL confocal microscope (Leica, Amsterdam, The Netherlands). Galectin-3 and NDP52 colocalization was visualized using a Dragonfly spinning-disk confocal microscope (Andor Technologies, Belfast, UK) equipped with 405, 488, 561 and 640nm lasers and a Zyla 4.2 sCMOS camera.
Sp8 wll confocal microscope
Manufactured by Leica
Sourced in Germany
The SP8 WLL confocal microscope is a high-performance imaging system designed for advanced biological and materials science research. The instrument utilizes a white light laser source to provide a broad range of excitation wavelengths, enabling the study of a wide variety of fluorescent samples. The system's confocal architecture allows for optical sectioning and high-resolution three-dimensional imaging.
Automatically generated - may contain errors
Lab products found in correlation
A1r lscm, by Leica (2 mentions)
Phalloidin alexa488, by Thermo Fisher Scientific (1 mentions)
Lipidtox green, by Thermo Fisher Scientific (1 mentions)
Filipin complex from streptomyces filipinensis, by Merck Group (1 mentions)
Application suite x las x, by Leica (1 mentions)
Cell tak, by Corning (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Lysotracker green, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
9 protocols using sp8 wll confocal microscope
1
Confocal Microscopy of Macrophage Lysosomes
2
Confocal Microscopy for Bacterial Labeling
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Confocal microscopy was used to
confirm the in vitro accumulation of Cy50.5CD9PIBMA39 on UBI29–41-Ad2 labeled bacteria. 0.2 mL of 25 mM Na-NH4-acetate
buffer pH 5 solution containing 1 × 108 CFUs S. aureus, 10 μM UBI29–41-Ad2, and 10 μM Hoechst 33342 was incubated for 30 min in
a dark shaking water bath at 37 °C. Samples were washed twice
in PBS as described above, after which they were incubated in 0.2
mL of a 1 μM solution of Cy50.5CD9PIBMA39 for 60 min in a dark shaking water bath at 37 °C. Samples
were washed thrice in PBS as described above, and finally resuspended
in 100 μL PBS. Ten microliters of labeled bacteria were pipetted
onto culture dishes with glass inserts (ø35 mm glass-bottom dishes
No. 15, poly(d -lysine) coated, γ-irradiated, MatTek
Corporation). Images were acquired at intervals of 1, 4, and 24 h
in a single field of view using a Leica SP8 WLL confocal microscope
(λex 633 nm, λem 650–700 nm) under 100× magnification
using Leica Application Suite Software Suite 4.8. Pictures were loaded
in ImageJ software (ImageJ 1.44p, NIH, USA) to draw a profile line
over a single representative stained bacterium in the Cy5 and the
Hoechst spectrum to estimate the distribution of both dyes in the
bacterial membrane and cytoplasm.
confirm the in vitro accumulation of Cy50.5CD9PIBMA39 on UBI29–41-Ad2 labeled bacteria. 0.2 mL of 25 mM Na-NH4-acetate
buffer pH 5 solution containing 1 × 108 CFUs S. aureus, 10 μM UBI29–41-Ad2, and 10 μM Hoechst 33342 was incubated for 30 min in
a dark shaking water bath at 37 °C. Samples were washed twice
in PBS as described above, after which they were incubated in 0.2
mL of a 1 μM solution of Cy50.5CD9PIBMA39 for 60 min in a dark shaking water bath at 37 °C. Samples
were washed thrice in PBS as described above, and finally resuspended
in 100 μL PBS. Ten microliters of labeled bacteria were pipetted
onto culture dishes with glass inserts (ø35 mm glass-bottom dishes
No. 15, poly(
Corporation). Images were acquired at intervals of 1, 4, and 24 h
in a single field of view using a Leica SP8 WLL confocal microscope
(λex 633 nm, λem 650–700 nm) under 100× magnification
using Leica Application Suite Software Suite 4.8. Pictures were loaded
in ImageJ software (ImageJ 1.44p, NIH, USA) to draw a profile line
over a single representative stained bacterium in the Cy5 and the
Hoechst spectrum to estimate the distribution of both dyes in the
bacterial membrane and cytoplasm.
3
Confocal Imaging of Midgut Samples
4
Comparative Embryogenesis Timing Analysis
5
Histochemical Analysis of Intestinal and Liver Tissues
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The small intestine was embedded in paraffin after fixing in 4% paraformaldehyde. Tissue was cut into 3.5-μm sections and stained with hematoxylin and eosin (H&E) solution. H&E staining was observed under an Olympus BX51 microscope; for frozen sections of liver and small intestine, tissues were fixed in 4%paraformaldehyde followed by 30% sucrose overnight and then embedded in optimal cutting temperature compound at −80°C. Tissues were cut into 8-μm sections to observe GFP-VSV signal in liver or for immunofluorescence staining, and images were acquired under a Leica SP8 WLL confocal microscope.
6
Immunofluorescence Imaging of HSC-4 Cells
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HSC-4 cells were grown overnight on confocal dish. Immunofuorescence was performed as described (22 (link)). The images were acquired in Nikon A1R LSCM or Leica SP8 Wll Confocal Microscope.
7
Western Blotting and Immunofluorescence Analysis
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Western blotting was performed as reported earlier [17] . Total proteins extracted from cells were resolved on SDS-PAGE, transferred onto nitrocellulose membranes, which were incubated with corresponding primary antibody followed by horseradish peroxidase-linked secondary antibody. The images were developed using Clarity western ECL substrate (Bio-Rad) and images were captured on X-Ray sheet.
Densitometric Analysis was performed using software ImageJ 1.52v. the average and standard deviation of fold change based on control from biological replicates were plotted .
Immuno uorescence staining and confocal microscopy HSC-4 cells were grown overnight on confocal dish. Immunofuorescence was performed as described [17] . The images were acquired in Nikon A1R LSCM or Leica SP8 Wll Confocal Microscope.
Densitometric Analysis was performed using software ImageJ 1.52v. the average and standard deviation of fold change based on control from biological replicates were plotted .
Immuno uorescence staining and confocal microscopy HSC-4 cells were grown overnight on confocal dish. Immunofuorescence was performed as described [17] . The images were acquired in Nikon A1R LSCM or Leica SP8 Wll Confocal Microscope.
8
Cellular Uptake and Lysosomal Localization of DOTA-αMSH-PEG-C' Dots
9
Cytocompatibility Evaluation of Scaffolds
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The cytocompatibility of the scaffolds was further assessed by a LIVE/DEAD staining. Cells on the scaffolds were stained with 10 µg/mL fluorescein diacetate (FDA) in PBS and 1 mg/mL Propidiumiodide in PBS at 3, 5, and 7 weeks. Based on intracellular esterase activity, viable cells convert FDA to green fluorescein, which stains viable cells green. As a nucleic acid dye, propidiumiodide can only enter compromised cell membranes of dying or dead cells, dyeing them red. Pictures were taken with a Leica SP8 WLL confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany). Non-quantifying experiments were confirmed with two biological donors.
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