Freshly extracted embryo populations were immediately fixed in cold 4% (w/v) paraformaldehyde and their eggshells were freeze-cracked by placing at −20 °C during 15 min. Then, embryos were pelleted via centrifugation (2 min., 2000 rpms, RT) and paraformaldehyde replaced by cold acetone for permeabilization (2 min, −20 °C) and, finally twice washed in M9. After fixation and washing, M9 was removed and replaced by a freshly prepared solution of phalloidin-AF594 (10:1000 (v/v), Molecular Probes) and Hoecsht33342 (2:5000 (v/v), Molecular Probes). Embryos were incubated overnight at RT under gentle agitation and then washed via two series of centrifugation (2 min. 2000 rpms) with M9. The supernatant was discarded and the pelleted embryos suspended in 2–3 drops of Prolong Gold Antifade reagent (Molecular Probes) and transferred by pipetting for mounting between glass slides using ProLong™ Antifade Gold Reagent (Invitrogen). Three-dimensional images were acquired with a Leica DMRE TCS SP2 AOBS confocal microscope (oil-immersion objective × 63, 1.4 NA), assembled, and reconstructed using Image J software.
Prolong gold antifade reagent
Manufactured by Thermo Fisher Scientific
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ProLong Gold antifade reagent is a water-based mounting medium designed to retard the photobleaching of fluorophores in fluorescence microscopy applications. It is formulated to provide long-term preservation of fluorescent signals in fixed and immunolabeled samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (274 mentions)
Hoechst 33342, by Thermo Fisher Scientific (122 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (121 mentions)
Triton x 100, by Merck Group (99 mentions)
Lsm 710 confocal microscope, by Zeiss (94 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (80 mentions)
Lsm 780 confocal microscope, by Zeiss (61 mentions)
Bovine serum albumin (bsa), by Merck Group (55 mentions)
Lsm 510 meta confocal microscope, by Zeiss (46 mentions)
Axio observer z1, by Zeiss (45 mentions)
Paraformaldehyde (pfa), by Merck Group (43 mentions)
Lsm 710, by Zeiss (41 mentions)
Lsm 700 confocal microscope, by Zeiss (38 mentions)
Lsm 880 confocal microscope, by Zeiss (33 mentions)
Lsm 510, by Zeiss (31 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (26 mentions)
Lsm 700, by Zeiss (25 mentions)
Hoechst 33258, by Thermo Fisher Scientific (25 mentions)
Lsm 780, by Zeiss (24 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (23 mentions)
2 662 protocols using prolong gold antifade reagent
1
Confocal Microscopy of Labeled Embryos
2
Immunofluorescent Localization of LC3 in Tissue Sections
3
Visualizing Inflammasome Activation
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B6, B6Nlrp1b+ and B6Nlrp1b+ASC−/− macrophages grown on coverslips were either left untreated (mock) or stimulated with LeTx for 30 min after which the active caspase-1 FAM-YVAD-cmk FLICA probe (Gentaur Molecular Products) was added for another 45 min. Cells were fixed and mounted on glass slides using ProLong Gold Antifade reagent containing DAPI (Life Technologies). Fluorescence micrographs were taken on an Olympus microscope using a × 20 objective lens. Quantification of specks was performed in three micrographs and the mean grey value was determined in 10 cells using ImageJ software. Alternatively, cells grown on coverslips were treated with LeTx for 60 min, fixed in 4% paraformaldehyde and then stained with antibodies against ASC (1/400, AG-25B-0006, Adipogen) and caspase-1 (1/200, AG-20B-0042-C100, Adipogen). Secundary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (1/250, A-11029 and A-11037, Invitrogen) were used. Slides were mounted in ProLong Gold Antifade reagent with Dapi (P36935, Invitrogen). Confocal micrographs were taken on an Olympus microscope using a × 60 objective lens. Quantification of specks was performed on 10 micrographs.
4
Immunofluorescence Staining of Proliferation and Lineage Markers
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Fixed cells were rinsed with PBS, permeabilized with a solution of 0.1% Triton-X100 and 0.1% sodium citrate, and incubated in a PBS blocking solution, containing 3% BSA (Sigma) and 0.1% Triton X-100 (Sigma), followed by a 2-h incubation at room temperature under agitation with a rabbit polyclonal anti-Ki67 1:200 (Abcam, France). After three rinses in PBS, the cells were incubated with a fluorescent green goat anti-rabbit Alexa Fluor 488 (Life Technologies, France) diluted at 1:400. The cells were then incubated 5 min with 0.5 g/ml DNA intercalating Hoechst (Life Technologies), before being rinsed and mounted using Prolong Gold Antifade reagent (Life Technologies). For MAP2 and nestin staining, the cells were immunolabeled as described above. The primary antibody solutions used were as follows: a polyclonal chicken anti-MAP2 (1/200, Abcam) and a monoclonal mouse anti-nestin (1/200, Abcam). After three rinses in PBS, the cells were incubated in a fluorescent goat red anti-rabbit Alexa Fluor 594 (Life Technologies, France) and green goat anti-mouse Alexa fluor 488 (Life Technologies, France) diluted at 1:400. The cells were then incubated 5 min at RT with 0.5 g/ml DNA intercalating Hoechst (Life Technologies) before being rinsed and mounted using Prolong Gold Antifade reagent (Life Technologies).
5
Immunostaining Protocol for Extracellular Traps
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For the staining of ETs samples were permeabilized for 5 min (0.5% Triton X-100; Invitrogen) and blocked for 20 min (blocking buffer with 3% normal donkey serum, 3% cold water fish gelatin, 1% BSA, and 0.05% Tween20 in PBS). Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1:500; Invitrogen). Staining with aqueous Hoechst 33342 (0.5 mg/ml) was performed for 10 min. After washing, all coverslips were embedded in ProLong® Gold antifade reagent (Invitrogen).
For the co-staining of ETs and neutrophil elastase within ETs, samples were prepared as described above. Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs and a rabbit anti-neutrophil elastase antibody (1:25 in blocking buffer, Abcam #AB1876 [Cambridge, UK]) in blocking buffer. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1: 500; Invitrogen) or a goat anti-rabbit Alexa 568-conjugated antibody (1: 500; Invitrogen). And stained aqueous Hoechst 33342 and embedded in ProLong® Gold antifade reagent as described above.
For the co-staining of ETs and neutrophil elastase within ETs, samples were prepared as described above. Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs and a rabbit anti-neutrophil elastase antibody (1:25 in blocking buffer, Abcam #AB1876 [Cambridge, UK]) in blocking buffer. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1: 500; Invitrogen) or a goat anti-rabbit Alexa 568-conjugated antibody (1: 500; Invitrogen). And stained aqueous Hoechst 33342 and embedded in ProLong® Gold antifade reagent as described above.
6
Visualizing PRRT2 Localization in Hek-Nav1.2 Cells
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Hek-NaV1.2 cells transfected with PRRT2-HA, PRRT2ΔC-HA, or PRRT2ΔN-HA constructs were live labeled by diluting primary antibodies (mouse anti-HA, 1:500, Millipore) in culture medium for 30 min at 37 °C/5% CO2 to detect surface epitopes, followed by fixation with 4% paraformaldehyde and incubation with Alexa Fluor-594 secondary antibodies. After several washes in phosphate-buffered saline (PBS), coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) containing 4′,6′-diamidino-2-phenylindole for nuclear staining. Hek-Nav1.2 cells transfected with PRRT2-HA, PRRT2ΔC-HA or PRRT2ΔN-HA constructs were fixed in 4% paraformaldehyde at room temperature for 20 min, washed in PBS, and blocked with 10% bovine serum albumin (BSA) for 20 min. Samples were sequentially incubated with mouse anti-pan-Nav (Sigma; 1:100 in 5% BSA) and rabbit anti-HA, (Invitrogen; 1:500 in 5% BSA) primary antibodies, followed by Alexa 564-conjugated or 488-conjugated secondary antibodies (Invitrogen; 1:200 in 5% BSA) at room temperature. After several washes in PBS, coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) containing 4′,6′-diamidino-2-phenylindole for nuclear staining.
7
Immunofluorescence Staining of Cells and Tissues
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Sc were cultured and fixed in 2% paraformaldehyde. The cells were permeabilized using 0.1% triton X-100 for 2 min followed by blocking with 3% BSA for 30 min. Primary antibody (Abcam, Cat. no. ab12132) was used at a dilution of 1:100 (overnight incubation, 4 °C). Secondary antibody alexafluor 488- goat anti-rabbit (Life Technologies, Cat. no. A-11008) was used at a dilution of 1:500 (45 min incubation, RT), Nuclei were stained using Hoechst 3342. The coverslips were mounted on glass slides using prolong gold Antifade reagent (Invitrogen). Cell imaging was done using Ri-2 Epi-flourescence microscope (Eclipse TiNikon).
Testes were fixed in 4% paraformaldehyde followed by paraffin block preparation and tissue sectioning as described above (Histological analysis). The tissue sections were permeabilized in 0.5% triton X-100 for 20 min followed by blocking with 2% normal goat serum for 30 min. Primary antibody (Abcam, Cat. no. ab12132) was used at a dilution of 1:100 (overnight incubation, 4 °C). Secondary antibody alexafluor 488-goat anti-rabbit (Life Technologies, Cat. no. A-11008) was used at a dilution of 1:500 (4-h incubation, RT), Nuclei were stained using Hoechst 3342. Coverslips were mounted over the tissue sections using prolong gold Antifade reagent (Invitrogen). Tissue imaging was performed using Ri-2 Epi-flourescence microscope (Eclipse Ti Nikon).
Testes were fixed in 4% paraformaldehyde followed by paraffin block preparation and tissue sectioning as described above (Histological analysis). The tissue sections were permeabilized in 0.5% triton X-100 for 20 min followed by blocking with 2% normal goat serum for 30 min. Primary antibody (Abcam, Cat. no. ab12132) was used at a dilution of 1:100 (overnight incubation, 4 °C). Secondary antibody alexafluor 488-goat anti-rabbit (Life Technologies, Cat. no. A-11008) was used at a dilution of 1:500 (4-h incubation, RT), Nuclei were stained using Hoechst 3342. Coverslips were mounted over the tissue sections using prolong gold Antifade reagent (Invitrogen). Tissue imaging was performed using Ri-2 Epi-flourescence microscope (Eclipse Ti Nikon).
8
Immunofluorescence Staining of Keratinocytes
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Immunofluorescence (IF) staining was used to detect keratinocyte proliferation and differentiation. For IF staining of primary keratinocytes, cells were briefly fixed with 4% paraformaldehyde (4% PFA) for 15 min, rinsed with phosphate-buffered solution (1×) (PBS, 1st Base, Singapore), blocked with 10% donkey serum (Sigma, USA) and incubated at 4°C overnight with primary antibodies. Slides were then rinsed and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
For IF staining of paraffin sections, standard dewaxing and rehydration steps were performed. Antigen retrieval was performed using citrate buffer pH6 (Novus Biologicals, USA) and 2100 Antigen Retriever (ProteoGenix, France), followed by Proteinase K (Sigma, USA) digestion for 15 min at room temperature. Sections were rinsed with PBS and blocked with donkey serum (Sigma, USA), and then incubated with primary antibodies overnight at 4°C. Next, the slides were rinsed in PBS-T (0.05% Tween-20, BioBasic, Singapore) and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, and then rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
For IF staining of paraffin sections, standard dewaxing and rehydration steps were performed. Antigen retrieval was performed using citrate buffer pH6 (Novus Biologicals, USA) and 2100 Antigen Retriever (ProteoGenix, France), followed by Proteinase K (Sigma, USA) digestion for 15 min at room temperature. Sections were rinsed with PBS and blocked with donkey serum (Sigma, USA), and then incubated with primary antibodies overnight at 4°C. Next, the slides were rinsed in PBS-T (0.05% Tween-20, BioBasic, Singapore) and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, and then rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
9
Immunofluorescent Staining of Spleen and Liver Tissue
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Frozen spleen sections (~12 μm thick) were acetone fixed and stained with mAbs to MMM (MOMA-1; Acris Antibodies), MZM (ERTR9; Bachem), T cell zone FRC (Alexa Fluor 488–conjugated podoplanin; gp38; eBioscience), CD3 (500A2; eBioscience), B220 (RA3-6B2; eBioscience) and F4/80 (BM8; eBioscience), and MAdCAM-1 biotin conjugated (Serotec). Fluorochrome-conjugated goat anti-rat antibodies were used for detection of purified antibodies whilst fluorochrome-conjugated, streptavidin-conjugated antibodies were used for the detection of biotin-conjugated antibodies. Sections were counterstained with DAPI, mounted in Pro-long Gold anti-fade reagent (Invitrogen) and visualized using a Carl Zeiss inverted LSM META 510 confocal microscope. For distributional analysis of VaDsred+ RTEs within infected livers, liver tissue was fixed in 4% paraformaldehyde (PFA) for two hours before overnight incubation in 30% sucrose. Tissue was embedded in Optimal Cutting Temperature (OCT) medium (Sakura) and frozen on dry ice. Confocal microscopy was performed on 8–10 μm frozen sections. Sections were counterstained with DAPI, mounted in Pro-long Gold anti-fade reagent (Invitrogen) and visualized using a Carl Zeiss inverted LSM META 510 confocal microscope.
10
Visualization of ORF2 and IRF3 in Huh7 Cells
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Huh7 cells transiently transfected with the FL ORF2 and 112-608 ORF2 plasmids were seeded on coverslips at 60% confluency. Cells were mounted in ProLong anti-fade gold reagent containing DAPI (ThermoFisher Scientific, United States). Cells were fixed with 2% paraformaldehyde in 1X PBS and ORF2 proteins were visualized with goat Anti-FLAG primary and anti-goat Alexa fluor 596 secondary antibodies.
For visualization of IRF3 translocation, pUNO Huh7, ORF2 Huh7, and 112-608 Huh7 stable cells were seeded on coverslips at 60% confluency and infected with JEV at 0.5 MOI. Cells were then fixed with 2% paraformaldehyde in 1X PBS. Rabbit anti-IRF3, mouse anti-JEV, and goat anti-FLAG antibodies were used as primary. Anti-rabbit Alexa-488 and an anti mouse Alexa-596 secondary antibodies were used. Cells were mounted in ProLong anti-fade gold reagent containing DAPI (ThermoFisher Scientific, United States). Slides were visualized using the Olympus FV3000 confocal microscope.
For visualization of IRF3 translocation, pUNO Huh7, ORF2 Huh7, and 112-608 Huh7 stable cells were seeded on coverslips at 60% confluency and infected with JEV at 0.5 MOI. Cells were then fixed with 2% paraformaldehyde in 1X PBS. Rabbit anti-IRF3, mouse anti-JEV, and goat anti-FLAG antibodies were used as primary. Anti-rabbit Alexa-488 and an anti mouse Alexa-596 secondary antibodies were used. Cells were mounted in ProLong anti-fade gold reagent containing DAPI (ThermoFisher Scientific, United States). Slides were visualized using the Olympus FV3000 confocal microscope.
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