To examine liposomes infiltration to the mucus, mucus and Cy5-labeled liposomes were imaged under confocal microscopy, with and without NAC treatment. Mucin from the porcine stomach was dissolved to a concentration of 43.75 mg/mL in PBS and gently shaken overnight in RT to form a disulfide bond and form mucus. The mixture was let to achieve relaxation for 24 h. 10 mg/mL NAC was dissolved in PBS for NAC treatment. 8 μL of synthetic mucus was dripped on microscope slide. 2 μL of 10 mg/mL NAC treatment was dripped on top. After 1 h of incubation, 2 μL liposome were placed on top of the described set. A coverslip was placed on top with spacers that ensured that the coverslip was evenly spaced. PBS solution used as control. Images were taken using Zeiss LSM 700 Confocal laser scanning microscopy, and were processed using Zen 3.4 Blue edition.
Lsm700 confocal laser scanning microscopy
Manufactured by Zeiss
Sourced in Germany
The LSM700 is a confocal laser scanning microscope developed by Zeiss. It utilizes a laser to scan a specimen and create high-resolution images. The LSM700 allows for the visualization and analysis of samples at the cellular and subcellular level.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Zen 3.4 blue edition, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Triton x 100, by Samchun (1 mentions)
Cryostat, by Leica (1 mentions)
Microscope slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Alexafluor 488 goat anti rabbit iggs, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Particle bombardment, by Bio-Rad (1 mentions)
Plan apochromat 63x 1.40 oil dic m27 objective, by Zeiss (1 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Evos fl auto cell imaging system, by Thermo Fisher Scientific (1 mentions)
Multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (1 mentions)
15 protocols using lsm700 confocal laser scanning microscopy
1
Mucus-Liposome Infiltration Imaging
2
Immunofluorescence Staining of Sciatic Nerves
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The sciatic nerves, LSCs, and TA muscles were fixed in 4% paraformaldehyde (Sigma) at 4 °C overnight. After being washed three times with 0.1 M PBS, the tissues were immersed in 0.1 M PBS containing 30% sucrose (Sigma) at 4 °C overnight, followed by cryopreservation in OCT compound (Sakura Tissue Tek). Samples were then cryosectioned using Cryostat (Leica). After 1 h incubation in blocking solution containing 2% BSA, 5% normal donkey serum (Jackson ImmunoResearch), and 0.1% Triton X-100 (Samchun), samples were treated with blocking solution containing primary antibodies for 1 h, and then with blocking solution containing secondary antibodies for 1 h. IgG Alexa Fluor (Invitrogen) was used as a secondary antibody. After mounting tissue sections on microscope slides (Fisher Scientific) with DAPI (Vectashield), immunofluorescence was observed using LSM 700 confocal laser scanning microscopy (Carl Zeiss).
3
Immunofluorescence Assay Protocol
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Cells for IFA were fixed with 4% paraformaldehyde for 10 min at R/T in (PBS) and then permeabilized using 0.1% Triton X-100 for 10 min at R/T. After blocking with 1% BSA in PBS for 30 min, cells were incubated with primary antibody in PBS containing 1% BSA for 2 h followed by incubation with secondary antibody for 1 h. The nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole) for 3 min at R/T. After washing with PBS, coverslips were mounted on microscope slides using the Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL), and examined under LSM700 confocal laser scanning microscopy (Zeiss). Images were processed with NIH Image/J to determine fluorescence intensity (FI).
4
Confocal Imaging of Mammalian Cells
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For confocal microscopy analysis, the adherent mammalian cells were grown on coverslips for 24 h. 4% Paraformaldehyde-fixed cells were blocked in HEPES 10 mM, 3% bovine serum albumin (Sigma-Aldrich, A9418), 0.3% Triton X-100 (Sigma-Aldrich, T8787) diluted in PBS (1 h at room temperature) and incubated with the indicated primary (4°C, overnight) and secondary AlexaFluor® 488 goat anti-rabbit IgGs (1:1000; Molecular probes/Invitrogen, A11008; room temperature, 1 h) antibodies. Antibodies are listed in above section. Nuclei were counterstained with Hoechst 33,342 (Molecular Probes/Invitrogen, H3570). Samples were mounted with Vectashield® Antifade Mounting Medium (Vector Laboratories, H-1900) and analyzed with Zeiss LSM700 confocal laser scanning microscopy (Oberkochen, Germany).
5
Subcellular Localization of ThNAC7
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The coding sequence (CDS) of ThNAC7 with no termination codon was fused with the C-terminus of the GFP following the CaMV 35S promoter. 35S::GFP was used as control. All primer used are listed in Table S1 . The 35S::ThNAC7-GFP and 35S::GFP plasmids was respectively imported into onion epidermal cells by particle bombardment (Bio-Rad, Hercules, CA, USA). The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (10 μg·mL−1) with phosphate buffered saline for 5 min. The transformed onion epidermal cells were visualised by LSM700 confocal laser scanning microscopy (Zeiss, Jena, Germany).
6
Immunofluorescence Analysis of Myospryn and Desmin Colocalization
7
Immunofluorescence Staining of Cell Lines
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Fibroblasts and Neuro2A cells were treated as previously described.28 (link) Briefly, cells were fixed with paraformaldehyde at 4% for 30 min at 37°C and then permeabilized using a solution of 10% Triton X-100 diluted 1:5000 in PBS. After 1 h of blocking, cells were incubated with the primary antibody for 40 min at room temperature and, after washes, with the secondary antibody for 20 min at room temperature. Coverslips were mounted using Mowiol solution. Images were acquired using Zeiss LSM700 confocal laser scanning microscopy (Zeiss, Oberkochen, Germany) or the EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) and cells with aggregates were manually counted.
8
RNA Scope FISH on Rat Liver Tissue
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FISH targeting PICALM-AU1 in rat liver tissue sections was performed using a commercially available RNA scope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, USA) by following the manufacturer's instruction. Fluorescent IHC staining target PICALM-AU1 was performed after FISH staining as described in the above section (Histopathology, Masson's Trichrome staining, and immunohistochemistry). Zeiss LSM 700 confocal laser scanning microscopy were used to visualize FISH results (Carl Zeiss, Germany).
9
Whole-Brain Immunohistochemistry with Confocal Microscopy
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Whole-brain immunohistochemistry was performed as described (66 (link)) with some modifications in SI Appendix . Fluorescent images were captured with TCS-SP5 laser-scanning confocal microscopy (Leica) with a HC PL Fluotar 10×/0.30 objective (catalog no. 506505, Leica) or HC PL APO 20×/0.70 objective (catalog no. 506513, Leica) and the Application Suite Advanced Fluorescence software (Leica) or LSM700 laser-scanning confocal microscopy (Carl Zeiss) with a Plan-APOCHROMAT 10×/0.45 or 20×/0.8 objective (Carl Zeiss) and ZEN 2011 software.
10
Tracking Amyloid Precursor Protein Trafficking
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Sub-confluent 7WD10 cells in 35-mm glass-bottom dishes were treated with mouse monoclonal anti-human APP antibody 6E10 (1:250 dilution) and biotinylated LME-tet (50 µM) on ice for 30 min. After washing, cells were incubated at 37 °C for the indicated times, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.2% TritonX-100. APP was detected using Alexa Fluor 546-conjugated goat anti-mouse IgG antibody. LME-tet was detected using Alexa Fluor 488-conjugated streptavidin. Early endosome antigen 1 (EEA1) was detected using rabbit polyclonal anti-EEA1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. Lysosome-associated membrane glycoprotein 1 (LAMP1) was detected using rabbit polyclonal anti-LAMP1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. For detection of the intracellular acidic compartment, cells were treated with 75 nM Lysotracker DND-99 (Molecular Probes, Eugene, OR, USA) for 1 h, followed by PFA fixation and permeabilization with 500 µg/ml digitonin (Wako Chemicals). Fluorescent images were analyzed using LSM700 laser scanning confocal microscopy (Zeiss, Oberkochen, Germany) and Image J software (NIH).
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