For fluorescent immunocytochemistry, the SACC-LM cells were cultured in 24-well cell culture plates for 24 h at 37°C in a humidified atmosphere containing 5% CO2. Then cells were fixed for 20 min with 4% paraformaldehyde and were permeabilized with PBS containing 0.2% Triton X-100 for 15 min. Normal goat serum working fluid incubated for 30 min at 37°C and incubated with primary antibody overnight at 4°C, and then staining was detected with fluorescein-conjugated secondary antibodies (PeproTech; 1:200) for 1 h at room temperature in dark condition. After washing with PBS for 3 times, nuclear was stained in 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. Immunofluorescence signals were examined using a fluorescence microscope (Leica, Germany). SACC tissues were cut into 8-μm thick sections, kept at −80°C until use. Sections were permeabilized with 0.5% Triton X-100 and pre-blocked with normal goat serum working fluid at 37°C for 30 min. Anti-Cathepsin D rabbit antibody (ab75852, 1:200, Abcam) was incubated, and sections were visualized after fluorescein-conjugated secondary antibodies and DAPI were stained.
Fluorescein conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescein-conjugated secondary antibodies are laboratory reagents used for immunodetection. They contain fluorescein, a fluorescent dye, covalently attached to the secondary antibody. These antibodies can bind to and fluorescently label target proteins or molecules, enabling their visualization and detection in various experimental techniques.
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Lab products found in correlation
Fluorescence microscope, by Olympus (8 mentions)
Lsm 780, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Biosesang (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fluorescein conjugated secondary antibody, by Abcam (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Ficoll density gradient centrifugation, by Merck Group (1 mentions)
Collagen, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
Endothelial cell growth medium, by Lonza (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Anti pecam1, by BD (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axioplan imager microscope, by Zeiss (1 mentions)
Ab21027, by Abcam (1 mentions)
32 protocols using fluorescein conjugated secondary antibody
1
Fluorescent Immunocytochemistry and Immunohistochemistry
2
Immunofluorescence analysis of OSCC cells
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The OSCC cells were cultured in 24-well cell culture plates, fixed for 15 min with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 min. After blocking with normal goat serum working fluid for 1 h at 37 °C, primary antibody was incubated overnight at 4 °C, and then staining was detected with fluorescein-conjugated secondary antibodies (PeproTech; 1:200) for 1 h in dark condition. Finally, cells were stained with 4,6-diamidino-2-phenylindole (DAPI; blue) to show the nuclear position for 5 min. Immunofluorescence signals were examined using a fluorescence microscope (Leica, Bensheim, Germany).
3
Immunofluorescence Staining of HA Hydrogels
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The HA hydrogels were washed three times with DPBS, and the washed gels were fixed for an hour at room temperature with 4 % paraformaldehyde (Biosesang, Korea). The fixed hydrogels were permeabilized with 0.3 % (v/v) Triton X-100 in DPBS (PBS-T) at room temperature for 30 min. After blocking with 1 % (w/v) BSA in PBS-T for an hour at room temperature, the hydrogels were incubated in the primary antibody solution (1:200) overnight at 4 °C. The samples were then washed with DPBS and incubated in fluorescein-conjugated secondary antibodies (Thermo Scientific) diluted 1:200 in 1 % BSA in PBS-T for 2 h at room temperature under dark conditions. For F-actin staining, Texas red-X phalloidin (Thermo Scientific) was used. The unbound antibodies were washed with DPBS, and samples were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Thermo Scientific) to observe the cellular nuclei. The fluorescence images were observed using Cytation3.
4
Isolation and Characterization of Circulating Endothelial Progenitor Cells
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Circulating EPC were isolated and cultured from peripheral mononuclear cells obtained from rats according to methods described previously 17 . Mononuclear cells were separated by Ficoll density gradient centrifugation (Sigma-Aldrich), and 5 × 106 cells were seeded into 6-well culture plates coated with collagen (5 μg/cm2, Sigma-Aldrich). The cells were cultured in endothelial cell growth medium (Lonza Ltd., Basel, Switzerland). The appearance of colony-forming cells with well-circumscribed monolayers of cobblestone-like cells was identified and recorded using a phase-contrast inverted microscope (Leica Biosystems, Wetzlar, Germany). The cultured endothelial cells were seeded into 24-well plates (Corning Inc., Corning, NY, USA) coated with 70 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), at a density of 5 × 105 cells per well, and were incubated at 37 °C. Tube formation of endothelial cells during 24-72 h after cell seeding was observed and photographed under an inverted microscope. The cultured endothelial cells and capillary-like cell formations were stained with CD31 antibody (Novus International, St. Louis, MO, USA) and fluorescein-conjugated secondary antibodies (Thermo Fisher), and calcein-AM in living cells (Sigma-Aldrich).
5
PECAM1 Staining of Mouse Cardiac Vasculature
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E12.5 mouse embryos or the hearts from P1 pups were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) overnight at 4 °C, and then transferred into 30% sucrose overnight at 4 °C. The tissues were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) and sectioned. The frozen sections were placed in room temperature for 30 min, blocked with 5% donkey serum, 0.5% Triton X-100 in 1 × PBS for one hour at room temperature. The sections were stained with anti-PECAM1 (BD Biosciences, New Jersey, USA) overnight at 4 °C and then stained with fluorescein-conjugated secondary antibodies (Life Technologies, Waltham, MA, USA) for 1 h at room temperature. Sections were visualized using a fluorescence microscope-Axio Imager Z2 (ZEISS, Oberkochen, Germany). All sections were imaged using a × 20 objective. The ImageJ software was used for image analysis. The ventricle areas of the hearts were imaged and analyzed. Vascular density was calculated as PECAM1+ pixels/total field pixels.
6
Immunostaining of Retinal Cryostat Sections
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Immunostaining of retinal cryostat sections were performed as described previously. (Patel et al., 2016 (link); Shosha et al., 2016 (link)) Eyes were enucleated, fixed in 4% paraformaldehyde for overnight at 4°C, washed in PBS, and cryoprotected in 30% sucrose. Cryostat sections (10 um) were obtained and mounted on glass slides. The sections were permeabilized in 0.05% Triton X-100 for 10 min and blocked in 10% normal goat serum containing 1% BSA for 1 h at room temperature. Followed by blocking, the sections were incubated with primary antibodies [SMO, Synaptophysin, Protein kinase Cα (PKCα), choline acetyl transferase (ChAT), Calbindin and Glial fibillary acidic protein (GFAP)] overnight at 4°C. Next day, they were washed and further incubated with Fluorescein-conjugated secondary antibodies (Life Technologies, Grand Island, NY, United States) for 1 h at room temperature. Finally, they were washed in PBS and covered with mounting medium and DAPI (Vectashield Vector Laboratories, Burlingame, CA, United States). Images of the stained section were obtained using a Zeiss (Thornwood, NY, United States) Axioplan Imager microscope and Zeiss Axiovision 4.8.2 software.
7
Immunofluorescence Staining for Sema3A, CD31, and α-SMA
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Immunofluorescence was performed as previously described [18 (link)]. The paraffin-embedded sections were deparaffinized with xylene and treated with heat-mediated antigen unmasking solution. Then, the sections were permeabilized in 0.25% Triton X-100 for 15 min. After being blocked with 20% donkey serum for 30 min., the sections were incubated with the primary antibodies anti-Sema3A antibody (1:100, ab199475, Abcam), anti-CD31 antibody (1:50, AF3628, R&D Systems, RRID: AB_2161028 ), anti-α-SMA antibody (1:100, ab5694, Abcam, RRID: AB_2223021 ) and anti-α-SMA antibody (1:100, ab21027, Abcam, RRID: AB_1951138 ) at 4 °C overnight, then incubated with the corresponding fluorescein-conjugated secondary antibodies (1:100 dilution; Life Technologies). Nuclei were stained with DAPI for 10 min. All sections were observed under a confocal microscope (Nikon, Tokyo, Japan).
8
Protein Expression and Quantification
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LM strips and cultured SMCs were homogenized in the lysis buffer containing protease and phosphatase inhibitor cocktails (ThermoFisher Scientific) and the amount of protein was measured using a RC-DC-protein assay kit (BioRad). Samples were separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Bedford, MA), and probed with primary antibodies and finally, with fluorescein-conjugated secondary antibodies (Life Technologies). The bands were visualized using an enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL). Phosphorylation of serine 19 in myosin light chain 2 (p-MLC) was evaluated by calculating the ratio of the band intensity of p-MLC to that of t-MLC, which was quantified on a separate immunoblot in which the same amount of sample was loaded (Figure 5G ). For the experiments shown in Figures 3B and 7B , an additional normalization of saline- and cytokine-treated samples to non-treated naïve samples (Control) was performed. This additional normalization was required because the basal levels of p-MLC/t-MLC fluctuated among the individual mice and/or separate experiments.
9
Immunofluorescence Characterization of Stem Cells
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hUDSCs or hADSCs were cultured on coverslips, washed with PBS and fixed with 4% paraformaldehyde (Biosesang, Korea) for 10 minutes at room temperature. Following fixation, the cells were washed with PBS and then blocked with PBS, containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100, for 30 minutes at room temperature, and incubated overnight with anti-vimentin, anti-E-cadherin, or anti-oct4 (Santa Cruz Biotechnology, Inc., USA) antibodies in a humidified chamber at 4°C. The cells were then incubated with fluorescein-conjugated secondary antibodies (Life technology, USA) and 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA) for 1 h, in the dark, at room temperature. Following this, the coverslips were mounted with fluorescence mounting medium (DAKO, CA, USA) and cell images were observed under a confocal microscope (Zeiss, Germany).
10
Immunofluorescence Analysis of LG Inflammasome Pathways
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The paraffin sections of LGs (4 µm) were stained with H&E and subsequently observed with a microscope. The LG cryosections or primary LG epithelial cells were fixed with 4% paraformaldehyde (Solarbio, Wuhan, China). The LG cryosections were incubated with NLRP3 (1:200; Affinity Biosciences, Cincinnati, OH, USA), caspase-1 (1:200; ABclonal, Wuhan, China), and GSDMD (1:200; Affinity Biosciences), and the primary LG epithelial cells were incubated with AQP5 (1:200; Abcam, Cambridge, MA, USA), epithelial cell adhesion molecule (1:200; Affinity Biosciences,), NLRP3 (1:200; Affinity Biosciences), caspase-1 (1:200; ABclonal), and GSDMD (1:200; Affinity Biosciences), followed by fluorescein-conjugated secondary antibodies (1:200; Life Technologies, Grand Island, NY). The cryosections were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).
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