The lung sections were washed using phosphate-buffered saline after deparaffinization and rehydration. The treated MH-S cells were fixed in 4% formaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100 for 20 min. The sections and cells were blocked with 5% bovine serum albumin at room temperature. The samples were incubated with primary antibodies against TFEB, LC3, p62, LAMP1, ubiquitin, or F4/80 overnight at 4 °C followed by incubation with secondary antibodies for 2 h at room temperature in the dark. After washing, the cells were mounted with ProLong® Diamond Antifade Mountant, containing DAPI (ThermoFisher, Waltham, MA, USA). Images were collected by fluorescence microscopy (Nikon, Tokyo, Japan).
Prolong diamond antifade mountant containing dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Diamond antifade mountant is a ready-to-use solution that contains DAPI, a fluorescent dye that binds to DNA. It is designed to preserve fluorescent signals and reduce photobleaching in fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8 confocal microscope, by Leica (5 mentions)
Opal multiplex kit, by PerkinElmer (4 mentions)
8 well chamber slide, by BD (3 mentions)
Lsm800, by Leica (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Tcs sp8, by Leica (2 mentions)
Axio zoom v16, by Zeiss (2 mentions)
C2 confocal microscope system, by Nikon (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Sp8 uv confocal microscope, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
Prolong diamond antifade mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Tmrdirect ligand, by Promega (1 mentions)
Nunc lab tek 2 8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Fv1000d, by Olympus (1 mentions)
Saponin, by BD (1 mentions)
Blocking buffer, by Agilent Technologies (1 mentions)
G2654, by Merck Group (1 mentions)
33 protocols using prolong diamond antifade mountant containing dapi
1
Immunofluorescence Analysis of Lysosomal Proteins
2
Immunofluorescence Imaging of Organoids
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Patient-derived organoids were cultured in 8-well chamber slides (BD Biosciences, East Rutherford, NJ, USA), fixed in 4% PFA for 30 min, and washed with PBS twice. We used whole-mount blocking buffer (WBB, containing 5% FBS, 0.2% BSA, 0.3% Triton X-100 in PBS) for blocking and permeabilizing the samples for 30 min. Samples were incubated with primary antibodies at 4 °C overnight in WBB, they were washed with PBS, labeled secondary antibodies were applied overnight at 4 °C and the organoids were mounted with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific). We used a Leica TCS SP8 confocal microscope for imaging and the ImageJ software for analysis and quantification. The antibodies used are listed in Table S1 .
3
Immunofluorescent Staining of Golgi Complex
4
Visualizing SARS-CoV-2 Spike Protein Binding
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ACE2-EGFP-expressing HEK293T cells (1.5 × 104 cells/well) were incubated at least 24 h before addition of S protein. In each well, the cells were incubated with S protein (50 nM) or HiLyte Fluor 555-labeled S protein (100 nM) for 3 h at 37 °C, 5% CO2. Cells were fixed in 4% paraformaldehyde for 10 min to stop the reaction and mounted with ProLong Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Imaging was performed on a BZ-X800 microscope (Keyence, Osaka, Japan). ACE2-internalized cells were counted in each section and normalized without S protein as a control.
5
Immunofluorescence analysis of desmosome proteins
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HaCaT cell line was seeded on glass coverslips with cell culture chambers (Nun Lab-Tek II Chamber Slide System, Thermo Fisher Scientific, Waltham, MA, USA) and cultured for at least 2 days in DMEM GlutaMAX medium containing 1 mM CaCl2 and 10% FCS and grown to confluence. Cells were treated with either IgG from PV patients’ or HD serum for 20 h in DMEM GlutaMAX medium containing 1 mM CaCl2 without FCS. After removing the medium and washing with PBS 1× complemented with CaCl2 and MgCl2 (Eurobio Scientific, Les Ulis, France), the cells were then fixed with ethanol 100% for 10 min at RT. The fixed cells were rinsed and permeabilized with Triton X-100 at 0.3% for 10 min (Sigma). After washing, cells were blocked for 30 min with 1% rat serum in PBS 1× at RT. Fluorescent-labeled antibodies staining DSG3 and flotillin-2, a protein associated with lipid microdomains that interact with desmosome proteins [28 (link)], were incubated for 1.5 h in the dark at RT in PBS 1× containing 1% BSA. Finally, coverslips were mounted with ProLong™ Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific, Waltham, MA, USA). All samples were analyzed with a Leica SP8-UV confocal microscope.
6
Immunostaining of Cells and Organoids
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Cells were fixed in 4% paraformaldehyde (PFA) for 20 min, washed with PBS, and then blocked and permeabilized in blocking buffer (PBS + 5% FBS + 0.2% BSA + 0.3% Triton X-100). Primary antibodies were applied at 4 °C overnight and then secondary antibodies for 2 h at room temperature (RT) (all diluted in blocking buffer). Samples were covered with ProLong Diamond antifade mounting medium with DAPI (Thermo Fisher Scientific).
Organoids were cultured in 8-well chamber slides (BD Biosciences, East Rutherford, NJ, USA), fixed in 4% PFA for 40 min, washed with PBS three times, and blocked and permeabilized with WBB whole-mount blocking buffer (PBS + 5% FBS + 0.2% BSA + 0.3% Triton X-100) for 2 h. Samples were incubated with primary antibodies at 4 °C overnight in WBB, and labelled with secondary antibodies for 2 h at RT. Organoids were then mounted with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific). Confocal images were taken with a Leica TCS SP8 microscope, and we used the ImageJ software for analysis and quantification. Antibodies are listed inTable S3 .
Organoids were cultured in 8-well chamber slides (BD Biosciences, East Rutherford, NJ, USA), fixed in 4% PFA for 40 min, washed with PBS three times, and blocked and permeabilized with WBB whole-mount blocking buffer (PBS + 5% FBS + 0.2% BSA + 0.3% Triton X-100) for 2 h. Samples were incubated with primary antibodies at 4 °C overnight in WBB, and labelled with secondary antibodies for 2 h at RT. Organoids were then mounted with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific). Confocal images were taken with a Leica TCS SP8 microscope, and we used the ImageJ software for analysis and quantification. Antibodies are listed in
7
Maspin Protein Localization in Cells
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Cells were plated in a Nunc Lab-Tek II 8-well chamber slide (Thermo Fisher Scientific) and were allowed to adhere overnight. Cells were washed two times with phosphate-buffer saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Fixed cells were washed with PBS and permeabilized with 100% ice-cold methanol for 10 min at − 20 °C. Samples were treated with 0.2% Triton-X in PBS for 10 min at RT. After blocking with 10% goat serum in PBS for 60 min at RT, samples were incubated with anti-maspin antibody (clone G167-70, 554292, BD Pharmingen, San Diego, CA, USA and clone EAW24, NCL-MASPIN, Leica Biosystems, Newcastle Ltd., UK) at 4 °C overnight, followed by incubation with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific). Cells transfected with the plasmid DNA expressing HaloTag-fused maspin were labeled with TMRDirect ligand (Promega) according to the manufacturer’s instructions. Slides were mounted by ProLong Diamond Antifade Mountant containing DAPI (Thermo Fisher Scientific). Confocal fluorescent images were obtained using confocal laser canning microscopy (FV1000D, Olympus, Tokyo, Japan).
8
Deparaffinization and Antigen Retrieval for Immunostaining
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PFA-fixed and paraffin-embedded sections were deparaffinized and rehydrated according to standard methods. Sections were then boiled in Tris-EDTA high-pH buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween-20 in distilled water, pH = 9.0) for 15 min and allowed to cool to RT for 20 min. After washing, sections were blocked in WBB, primary antibodies were applied at 4 °C overnight, and secondary antibodies were applied for 2 h at room temperature (all in WBB). Samples were covered with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher Scientific).
9
Immunofluorescence Staining of Extracellular Vesicles
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Cells were fixed in 4% PFA for 20 min, blocked, and permeabilized in blocking buffer (PBS with 0.1% BSA, 5% FBS, and 0.1% Triton X-100). Cells were then incubated with primary antibodies at 4 °C overnight and then in secondary antibodies in blocking buffer for 2 h at room temperature. After covering samples with ProLong Diamond antifade mountant containing DAPI (Thermo Fisher), images were taken with a Zeiss LSM800 or Leica TCS SP8 confocal microscope. In some experiments with the labelled medium-sized EV fraction, the blocking buffer contained 0.05% saponin (BD Biosciences) instead of Triton X-100. The antibodies used are listed in Table S2.
10
Quantitative Immunostaining of Murine Pancreas
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