Corneal buttons were de-epithelialized by 45 min incubation at 37 °C in EDTA (0.5 M in H2O, Sigma-Aldrich, Saint Louis, MO, diluted 25 times in PBS) and were fixed in acetone for 15 min at room temperature, followed by a rinse in Phosphate Buffer Saline (PBS). To prevent nonspecific staining, Fc receptor blocking antibody (anti-mouse CD16/CD32 purified, clone 93, eBioscience San Diego, CA) was used to block sections before staining. Corneas were incubated with antibodies at room temperature for 30–45 min. The following antibodies were used: Anti-Ia/Ib (MHC class II) (AF6-120.1), anti-CD45 (5C3), anti-CD86 (GL-1) from Biolegend (San Diego, CA) to stain corneal stromal APCs. Each step was followed by three thorough washings in PBS for 5–10 min. At the end samples were covered with mounting medium (Vector Laboratories) and analyzed by confocal laser scanning microscope (TCS 4D; Leica, Heidelberg, Germany). For histological evaluation, corneal sections were stained with H&E and examined using bright-filed microscopy.
Tcs 4d
Manufactured by Leica
Sourced in Germany, United States
The Leica TCS 4D is a confocal microscope system designed for high-resolution imaging. It provides advanced optical sectioning capabilities to capture detailed 3D and 4D (3D over time) images of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Ds 5mc camera, by Nikon (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Lsm image browser, by Zeiss (1 mentions)
Lsm510 meta software, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Las af lite software, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Dq bsa, by BD (1 mentions)
Dq bsa, by Leica (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest, by BD (1 mentions)
Dq green bsa, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsm 710, by Zeiss (1 mentions)
16 protocols using tcs 4d
1
Corneal Antigen-Presenting Cell Characterization
2
Cyclic Strain Induces Cytoskeletal Changes
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The cells were cultured in collagen I-coated, 6-well Bioflex plates containing serum-free medium for 24 h and then stimulated with cyclic strain for up to 1 day. At 6 h and 24 h, the cells were rinsed in phosphate-buffered saline (PBS) for 3 min, fixed in 5% paraformaldehyde (PFA) for 30 min, and permeabilized with 0.1% Triton in PBS for 10 min. Then, the cells were incubated with FITC-labeled phalloidin diluted at 1∶100 at room temperature. Finally, the cells were stained with DAPI for 5 min (1∶200 dilution) at room temperature. After being washed with PBS, cells were mounted with fluorSave reagent (Calbiochem) and analyzed by confocal microscopy (Leica TCS 4D). Three randomly selective microscopic fields were analyzed.
3
Fluorescent Microscopy Imaging Protocol
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Bright field and fluorescent specimens were observed under a Zeiss Axiovert 200 M. Filter set 09 (450–490 nm excitation, LP 515 nm) was used for GFP fluorescence. Filter set 14 (510–560 nm excitation, 590 nm emission) was used for detection of mRFP1 fluorescence. For Duolink analysis red signals (TX Red) were detected using filter set 31 (BP 565/30 nm excitation, BP 620/60 emission) and blue signals (UV) detected using filter set 49 (G365 nm excitation, BP 445/50 nm emission).
Samples were excited with UV-light produced by a HBO 50/Ac lamp and images were taken with a Nikon DS-5Mc camera. The software EclipseNet plug (Nikon) was used to measure and merge fluorescence images. CLSM was performed using either the confocal laser scanning module TCS 4D (Leica), or a Zeiss LSM510 META (Zeiss). For detection of GFP, specimens were excited using an Argon 488 nm laser, and the BP 505–550 filter was used for detection. For mRFP1 and PI fluorescence, a helium neon laser (543 nm) was used for excitation in combination with a LP 560 filter. For capture and processing of confocal images the Zeiss LSM 510 META software and the Zeiss LSM Image Browser version 3.5.0.359 was used.
Samples were excited with UV-light produced by a HBO 50/Ac lamp and images were taken with a Nikon DS-5Mc camera. The software EclipseNet plug (Nikon) was used to measure and merge fluorescence images. CLSM was performed using either the confocal laser scanning module TCS 4D (Leica), or a Zeiss LSM510 META (Zeiss). For detection of GFP, specimens were excited using an Argon 488 nm laser, and the BP 505–550 filter was used for detection. For mRFP1 and PI fluorescence, a helium neon laser (543 nm) was used for excitation in combination with a LP 560 filter. For capture and processing of confocal images the Zeiss LSM 510 META software and the Zeiss LSM Image Browser version 3.5.0.359 was used.
4
Immunofluorescence Analysis of FECD Specimens
5
Immunohistochemistry Protocol for Drosophila Tissue Analysis
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Immunohistochemistry was performed as previously described [45 (link)]. In brief: tissues were dissected and fixed in 4% paraformaldehyde for 20 min on ice. Afterwards they were washed in phosphate buffered saline (PBS) with 0.4% Triton X-100. Antisera were used in following concentrations: anti-LacZ (55976 Cappel/MP; 1:1000), anti-Rst (mAB 24A5; 1:10) [45 (link)], anti-Kirre (126i; 1:200) [46 (link)], anti-SNS (1:200) [47 (link)], anti-Hbs (AS-14; 1:400). The Hbs Antibody AS-14 was produced from a 14 Amino acid peptide AEPSNDDVYSKDDS (1083–1096) by Genscript Corporation using standard protocols. Confocal microscopy of the specimen was carried out with a Leica TCS4D. Comparative experiments were performed using the same laser intensities and by scanning all sets at the same day. Data was processed with AMIRA 5.2 (Indeed, Berlin, Germany) and Adobe Photoshop CS6. All images represent an N number of ≥10.
6
Confocal Imaging of Fluorescent Staining
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Imaging of fluorescence staining was done by confocal imaging of fixed cells with a laser-scanning confocal inverted microscope (Leica TCS 4D, Wetzlar, Germany), and a 63×/1.4 numerical aperture oil objective was used to image the samples. For indirect immunofluorescence studies, cells were grown on 100-mm2 coverslips, fixed in 3% paraformaldehyde and permeabilized (when necessary) with 0.2% Triton X-100. Coverslips were incubated in 5% bovine serum albumin (BSA) for 20 min and probed with primary antibodies (diluted 1:200 in PBS containing 5% BSA) for 2 h at room temperature. Cells were washed three times in PBS and incubated for 1 h at room temperature with Alexa Fluor Dyes [Alexa Fluor 488 goat anti-mouse IgG (H + L) (#A11001) and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (#A21071) both from Thermo-Fisher]. Coverslips were permanently mounted to the slides using fluorescent mounting medium (PROLONG-GOLD, Thermo Fisher Scientific).
7
Quantifying Autophagy Flux in Macrophages
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To quantify protein degradation induced by stimuli, autophagy flux was analyzed by flow cytometry and confocal microscopy using the self-quenched substrate DQ-Green BSA (Molecular Probes, Eugene, OR) as described in previous studies
[59 (link)]. Briefly, WT or Cat S-/- BMDMs were cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. WT BMDMs were in the presence or absence of Cat S inhibitor Z-FL-COCHO (10 μmol/L) cocultured with SL4 cells. WT or Cat S-/- BMDMs into the lower compartment were loaded with 10 μg/ml DQ-Green BSA for 15 min at 37°C. The cells were washed twice with PBS to remove excess label, then harvested at indicated time points. Cells were harvested, and Green-fluorescent of DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest (BD Biosciences) and FlowJo (Treestar) software. For confocal images analysis, cells were placed on coverslips after treatment as above and fixed with 4% formaldehyde. The fluorescent degradation products of DQ-BSA in lysosomes were imaged using a confocal laser-scanning microscope (TCS 4D; Leica, Heidelberg, Germany).
[59 (link)]. Briefly, WT or Cat S-/- BMDMs were cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. WT BMDMs were in the presence or absence of Cat S inhibitor Z-FL-COCHO (10 μmol/L) cocultured with SL4 cells. WT or Cat S-/- BMDMs into the lower compartment were loaded with 10 μg/ml DQ-Green BSA for 15 min at 37°C. The cells were washed twice with PBS to remove excess label, then harvested at indicated time points. Cells were harvested, and Green-fluorescent of DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest (BD Biosciences) and FlowJo (Treestar) software. For confocal images analysis, cells were placed on coverslips after treatment as above and fixed with 4% formaldehyde. The fluorescent degradation products of DQ-BSA in lysosomes were imaged using a confocal laser-scanning microscope (TCS 4D; Leica, Heidelberg, Germany).
8
Measuring Autophagic Flux in Macrophages
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Cat S-/- or WT BMDMs were transfected with mCherry-GFP-LC3 reporter construct by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as previously described in our lab
[31 (link)]. After transfection, Cat S-/- or WT BMDMs cocultured with SL4 cells without serum for 48 hrs. Cells were fixed with 4% paraformaldehyde and microphotographs of mCherry-GFP-LC3 fluorescence were obtained with the confocal laser-scanning microscope (TCS 4D; Leica, Heidelberg, Germany). Treatment-induced changes in GFP intensity in mCherry-positive puncta were calculated to depict autophagic flux.
[31 (link)]. After transfection, Cat S-/- or WT BMDMs cocultured with SL4 cells without serum for 48 hrs. Cells were fixed with 4% paraformaldehyde and microphotographs of mCherry-GFP-LC3 fluorescence were obtained with the confocal laser-scanning microscope (TCS 4D; Leica, Heidelberg, Germany). Treatment-induced changes in GFP intensity in mCherry-positive puncta were calculated to depict autophagic flux.
9
Autophagy Quantification with MDC
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MDC, an acid dye, serves as a specific marker for autophagic vacuoles, and thus employed for detection and quantification of autophagy. MDA-MB-468 and MCF-7 cells were seeded in 6-well plates at a concentration of 3 × 104 cells/well in a 5% CO2 incubator at 37°C for 24 h. Following transfection, 50 mM MDC (HY-D1027, MedChemExpress, NJ, USA) was added to each well for 15-min incubation, and then rinsed 3 times with PBS. The nuclei were then stained with DAPI. Finally, the fluorescence was visualized under a confocal laser-scanning microscope (TCS 4D; Leica, Heidelberg, Germany).
10
Paraffin-embedded cSCC Histology and Staining
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Paraffin-embedded SCC biopsies were provided from the pathological unit of the Department of Diagnostic, Clinic and Public Health Medicine of the University of Modena and Reggio Emilia. cSCC spheroids were fixed with 4% paraformaldehyde (PFA) for 30’ at RT and embedded in paraffin cSCC tissue or spheroids or skin reconstruct histology and staining (immunohistochemistry or immunofluorescence) were performed as previously described [12 (link)]. For all experiments, primary antibodies are indicated in Supplementary Table 2 . Micrographs were taken on a Confocal Scanning Laser Microscopy (Leica TCS4D, Leica, Exton, PA, USA).
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