OCT-embedded liver sections (4 μm thick) were mounted on a positively charged slide (SuperFrost® Plus, Menzel-Gläser; Thermo-Scientific, Waltham, MA, USA), incubated for 15 min with a blocking buffer (PBS; 0.2% Triton X-100; and 3% bovine serum albumin), and then incubated overnight at 4 °C with rabbit monoclonal AQP8 antibodies (1/100 dilution). Then, the samples were washed and incubated with a secondary Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 h. After washing the samples, nuclei were stained with DAPI (50 μM) for 10 min, and the coverslips were mounted with ProLong Gold. The samples were visualized using confocal microscopy (Nikon C1 Plus confocal microscope mounted on an Eclipse TE-2000-E2 inverted microscope), and the same microscope settings were used for imaging acquisition. No autofluorescence signals were detected in the samples incubated only with the primary or secondary antibody. In order to analyze the collected images, the same contrast/brightness adjustments were applied to every image within the set using the Fiji (ImageJ) software v. 2.9.0/1.53t. Transduction efficiency was calculated as previously described [14 (link)].
Alexa fluor 647 conjugated goat anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody is a fluorescent-labeled reagent used in immunoassays and other applications. It is designed to bind to rabbit primary antibodies, allowing for detection and visualization of target analytes.
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Lab products found in correlation
Syto 16, by Thermo Fisher Scientific (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Ab5032, by Merck Group (1 mentions)
Hoechst dna dye, by Thermo Fisher Scientific (1 mentions)
Mouse igg1κ isotype, by BioLegend (1 mentions)
Alexa fluor555 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
A1 confocal laser microscope system, by Nikon (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Anti met c12 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti ddk antibody, by OriGene (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Aposensor cell viability assay kit, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Anti type 1 collagen antibody, by Southern Biotech (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
12 protocols using alexa fluor 647 conjugated goat anti rabbit secondary antibody
1
Quantifying Hepatic AQP8 Expression
2
Immunocytochemical Staining of NCAM
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For immunocytochemical staining, cells were grown on glass coverslips and fixed with 4 % paraformaldehyde in PBS. Nonspecific binding was blocked with 1.5 % normal horse serum (Vector Laboratories, Burlingame, CA, USA) in PBS. Cells were labeled with 10 μg/ml of rabbit polyclonal anti-human NCAM antibody (AB5032; Millipore) followed by AlexaFluor 647 conjugated goat anti-rabbit secondary antibody (Molecular Probes) and inactive endosialidase-GFP fusion protein [33 (link)], all in PBS. Cover slips were mounted with ProLong Mounting Medium with DAPI (Molecular Probes). The staining was visualized with an Olympus BX50F-3 microscope and imaged by a PCO CCD Imaging SensiCam color camera and Image-Pro Plus 4.0 software.
3
Quantifying SARS-CoV-2 Infection by Immunofluorescence
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Fixed cells were washed three times with Dulbecco-modified PBS containing 0.2% BSA (DPBS/BSA), permeabilized with 0.1% Triton X-100 in DPBS/BSA and processed for immunodetection of viral N protein, automated fluorescence imaging, and image analysis. Briefly, viral NP was detected with an in-house-developed rabbit polyclonal antibody22 (link) counterstained with Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (ThermoFisher Scientific, catalogue number A32733); nuclear staining was done using Hoechst DNA dye (ThermoFisher Scientific, catalogue number H3570). Automated fluorescence imaging was done using a Molecular Devices Image-Xpress Nano high-content epifluorescence microscope equipped with a 10× objective and a 4.7-megapixel CMOS camera (pixel size, 0.332 μm). Image analysis was performed with CellProfiler-4 software (www.cellprofiler.org ). Automated detection of nuclei was performed using the Otsu algorithm inbuilt in the software. To automatically identify infected cells, an area surrounding each nucleus (5-pixel expansion of the nuclear area) was used to estimate the fluorescence intensity of the viral NP immunolabeled protein, using an intensity threshold such that <0.01% of ‘positive cells’ were detected in noninfected wells.
4
Immunohistochemical Profiling of Atherosclerosis
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Human endarterectomy segments were frozen in Tissue Tek (Sakura Finetek, Germany) and stored at − 80 °C. Plaques were cut into 6 µm thick serial sections and immobilised on microscope slides. Tissue sections were rehydrated and blocked with 10% normal goat serum (Vector Laboratories, USA). After blocking, the sections were incubated with mouse anti-human CD68 primary antibody (Dako, USA) and rabbit polyclonal P2Y12 primary antibody (Cat: GTX54796, GeneTex, USA) at 4 °C overnight. As controls, samples were stained with mouse IgG1κ isotype (BioLegend, USA), rabbit IgG isotype (Vector Laboratories) and only the primary antibody. Sections were subsequently incubated with an Alexa Fluor 555–conjugated goat anti-mouse secondary antibody (Thermo Fisher, USA) and an Alexa Fluor 647–conjugated goat anti-rabbit secondary antibody (Thermo Fisher, USA) for 30 min at RT. Nuclei were stained with DAPI. All images were captured using a Nikon A1 Confocal Laser Microscope System and processed using Fiji software.
5
Evaluating Erythroblast IGF2BP1 and HbF
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Erythroblasts (1 × 105 per condition) were treated with a Fix & Perm cell permeabilization kit (Invitrogen) according to the manufacturer’s instructions. Staining was performed during the permeabilization step. IGF2BP1 was detected with a monoclonal antibody (Cell Signaling Technology; clone D33A2, 1:100 dilution) and then Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (Life Technologies; 1:2,000 dilution). Cells treated with secondary antibody alone were used as a control. HbF was detected using phycoerythrin (PE)-conjugated mouse anti-human fetal hemoglobin monoclonal antibody (BD Pharmingen, San Jose, CA, USA; clone 2D12, 1:500 dilution); PE-conjugated mouse immunoglobulin G (IgG) (BD Pharmingen; 1:500 dilution) served as a control. Data were collected using the FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo v10.0 software. The percentage positive cells and geometric mean fluorescence intensity (MFI) was determined for each condition. In selected experiments, cells were simultaneously stained with antibodies to both IGF2BP1 and HbF and the percentage of double-labeled cells was determined.
6
Smad2 Activation and Collagen Dynamics
7
3D Histological Analysis of Tissue Samples
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For three-dimensional (3D) histological analysis, approximately 10 × 10 × 10 mm3 tissue blocks were fixed in 10% formaldehyde for 2 days and then washed in phosphate-buffered saline (PBS) for 4 days at 4 °C. Next, the specimens were cut into 350-μm-thick sections by using a vibratome. The primary antibodies anti-CD31 and anti-α-SMA were used to immunolabel the tissues. To detect the immunostained structures, Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody was used in combination with Alexa Fluor 546-conjugated goat anti-mouse or anti-rat secondary antibody (1:200, Invitrogen, Waltham, MA). The nuclei were stained using either propidium iodide or SYTO 16 (Invitrogen) at room temperature for 1 h.
8
Intracellular Staining for Lsd1 and FoxP3
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For intracellular Lsd1 staining, after cells were stained for surface markers, cells were fixed and permeabilized using an eBioscience FoxP3/Transcription Factor Staining Buffer Set (00-5523-00). Then cells were incubated with a rabbit anti-Lsd1 antibody (2139s; Cell Signaling Technology) at room temperature in the dark for 30 min, washed, then stained with an Alexa Fluor 647–conjugated goat anti-rabbit secondary antibody (A27040; Invitrogen) for 30 min in the dark at 4°C. A similar procedure was followed for FoxP3 intracellular staining, except that the FoxP3 antibody was directly fluorescent antibody conjugated (FJK-16s; eBioscience). For cell cycle analysis, after surface staining, fixation, and permeabilization, cells were stained with Ki-67 (561126; BD Biosciences) and DAPI (D21490; Invitrogen) on wet ice. Data were then acquired on an LSR II flow cytometer.
9
Immunohistochemical Analysis of Ano1 and c-kit in Tissue
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The fixed specimens were immersed in 2% Triton X-100 solution for 2 hours at 15°C for permeabilization. The primary antibodies used to reveal ICC were a monoclonal rabbit anti-c-kit antibody (cat. no. 1522–1; Epitomics, Burlingame, CA) and a polyclonal rabbit anti-Ano1 antibody (also known as anti-TMEM16A, cat. no. ab53212; Abcam, Cambridge, MA). A monoclonal mouse anti-CD31 antibody (cat. no. MS-353-S1; Thermo Scientific, Fremont, CA) was used to reveal the vasculature.
Before we applied the antibodies, the tissue was rinsed in PBS. This was followed by a blocking step, incubating the tissue with the blocking buffer (2% Triton X-100, 10% normal goat serum, and 0.02% sodium azide in PBS). The primary antibodies were then diluted in the dilution buffer (1:100, 0.25% Triton X-100, 1% normal goat serum, and 0.02% sodium azide in PBS) to replace the blocking buffer and incubated overnight at 15°C. Alexa-Fluor 647-conjugated goat anti-rabbit secondary antibody and Alexa-Fluor 546-conjugated goat anti-mouse secondary antibody (1:200; Invitrogen, Carlsbad, CA) were used to reveal the immunostained structures. Afterward, nuclear staining by SYTO16 (4 μmol/L; Invitrogen) was performed at 15°C for 1 hour. The labeled specimens were then immersed in the optical clearing solution FocusClear (CelExplorer, Hsinchu, Taiwan) before being imaged via confocal microscopy.
Before we applied the antibodies, the tissue was rinsed in PBS. This was followed by a blocking step, incubating the tissue with the blocking buffer (2% Triton X-100, 10% normal goat serum, and 0.02% sodium azide in PBS). The primary antibodies were then diluted in the dilution buffer (1:100, 0.25% Triton X-100, 1% normal goat serum, and 0.02% sodium azide in PBS) to replace the blocking buffer and incubated overnight at 15°C. Alexa-Fluor 647-conjugated goat anti-rabbit secondary antibody and Alexa-Fluor 546-conjugated goat anti-mouse secondary antibody (1:200; Invitrogen, Carlsbad, CA) were used to reveal the immunostained structures. Afterward, nuclear staining by SYTO16 (4 μmol/L; Invitrogen) was performed at 15°C for 1 hour. The labeled specimens were then immersed in the optical clearing solution FocusClear (CelExplorer, Hsinchu, Taiwan) before being imaged via confocal microscopy.
10
Evaluating γH2AX DNA Damage Response
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Cells were seeded into 24-well plates with poly-L-lysine-coated coverslips at a density of 5×10 4 cells/ml. Following treatment, cells on the coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing 3 times in PBS, the sections were permeabilized with 0.5% Triton X-100 in PBS for 30 min. Blocking was performed with 5% goat serum, followed by incubation of the slides with a rabbit polyclonal antibody against γH2AX (ab18311, 1:500 dilution, Abcam) at 4 °C overnight. The next day, after washing in PBS containing 0.5% Tween-20, the slides were incubated with an Alexa Fluor®647-conjugated goat anti-rabbit secondary antibody (1:100 dilution; Invitrogen Corp.) at 37 °C for 1 h. Additionally, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Beyotime) to label cell nuclei and were covered with a coverslip. The sections were then viewed using a Leica TCS4D confocal laser scanning microscope, and images were recorded digitally. Negative-control sections were treated in the same way, except that the primary antibodies were replaced with IgG
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