For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde for 15 min in PBS, permeabilized with 0.2% Triton X-100 in PBS, blocked with blocking buffer and then stained with primary antibodies against α-tubulin (ab52866, Abcam), α-actinin (A7811, Sigma-Aldrich), or cleaved caspase-1 p20 (PRS3459, Sigma-Aldrich) followed by Alexa Fluor 488/546/633 secondary antibodies. ASC was detected with an Alexa Fluor 488 Conjugated antibody (17507, Cell Signaling). Rhodamine phalloidin dye (R415, Invitrogen, Carlsbad, CA, USA) was incubated with the cells for 20 min at 37 °C for F-actin. The stained cells were visualized and analyzed using a laser scanning confocal microscope with a 63×/1.4NA oil immersion objective lens and excitation wavelengths of 488, 543, and 633 nm. All images were acquired using ZEN 2012 software.
Alexa fluor 488 conjugated antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488-conjugated antibody is a fluorescent-labeled secondary antibody. It is used to detect and visualize primary antibodies in various immunodetection techniques, such as immunofluorescence microscopy and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Ab52866, by Abcam (1 mentions)
A7811, by Merck Group (1 mentions)
Prs3459, by Merck Group (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
40 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated antibody, by Cell Signaling Technology (1 mentions)
Lsm 880 confocal fluorescence microscope, by Zeiss (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Eclipse ti u 100, by Nikon (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Millicell ez slide, by Merck Group (1 mentions)
Smad2 3 antibody d7g7 xp rabbit mab 8685, by Cell Signaling Technology (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Anti brdu antibody, by Bio-Rad (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
9 protocols using alexa fluor 488 conjugated antibody
1
Immunofluorescence Staining of Cell Structures
2
Rat AT-MSCs Differentiation Evaluation
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The undifferentiated and the fully differentiated rat AT-MSCs were cultured on two chambers-slide (Nunc, Rochester, NY, USA). Initially, fixation of the cells was occurred by using 4% paraformaldehyde for 10 min, followed by permeabilization of the cells that occurred with 100% methanol for 10 min. Blocking of the cells by using 5% goat serum was then performed for 1 h at room temperature (RT). Afterward, the cells were overnight incubated at 4 °C with the primary antibodies [mouse monoclonal anti-rat insulin Antibody 1/200 (Novus Biologicals, Littleton, CO, USA) and rabbit polyclonal anti-rat c-peptide antibody 1/100 (Cell Signaling Technology)]. The cells were kept for 2 h at room temperature with the secondary antibodies [1/200 anti-mouse immunoglobulin G (IgG; HL) Alexa Fluor 488 conjugated antibody and 1/100 anti-rabbit (IgG; HL) Alexa Fluor 555 conjugated antibody (Cell Signaling Technology)] after being washed with PBS. The stain [40, 6-diamidino-2-phenylindole (DAPI, Invitrogen, UK)] was used to counterstain the nuclei. No primary antibodies were utilized for the negative controls. The confocal images were taken finally by the Leica TCS SP8 microscope (Leica Microsystems, Mannheim, Germany).
3
Immunofluorescence Analysis of Chondrocyte Exosome Uptake
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For immunofluorescence, after reaching more than 80% confluence, chondrocytes were transferred to serum-free culture medium for 24 h and then incubated in complete culture medium with various exosomes for an additional three hours. The cells were then washed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min. The fixed cells were permeabilised with 0.1% Triton X-100 for 5 min and washed with PBS three times. Then, the cells were treated with primary antibodies overnight at 4°C in PBS supplemented with 1% bovine serum albumin (BSA). Cells were washed a further three times, then the secondary Alexa Fluor® 488-conjugated antibody (Cell Signalling Technology) was added and incubated for 1 h at room temperature. After washing using PBS, cells were mounted in ProLong® Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) and randomly-selected fields were imaged using an LSM-880 confocal fluorescence microscope (Carl Zeiss, Jena, Germany).
4
Immunofluorescence Analysis of Activated Neutrophils
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Freshly isolated human or murine bone marrow-derived neutrophils (1.5 × 105) were seeded on polylysine-coated glass coverslips (Ø 18 mm), allowed to settle for 30 min, and then treated with 100 nM PMA. After 3.5 h of activation, cells were fixed with 4% paraformaldehyde (PFA), blocked with 5% normal goat serum (Cell Signaling Technology) and 1% bovine serum albumin (BSA) (Carl Roth) and incubated with polyclonal antibodies against myeloperoxidase (MPO) (Abcam) for 2 h at room temperature. Cells were further incubated with a secondary anti-rabbit Alexa Fluor 488-conjugated antibody (Cell Signaling Technology) for 1 h, counterstained with DAPI and mounted in Dako fluorescent mounting medium (Dako). Cells were examined with an inverted microscope (Eclipse Ti-U 100, Nikon).
5
Analyzing Smad2/3 Activation in Gingival Fibroblasts
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Gingival fibroblasts seeded onto Millicell EZ slides (Merck KGaA, Darmstadt, Germany) were serum-starved overnight before being exposed to the indicated cell lysates with and without SB431542 for 30 min. Then, paraformaldehyde fixation blocking was conducted with 5% bovine serum albumin and 0.3% Triton X-100 in PBS. After permeabilization with 0.1% Triton X-100, cells were incubated with Smad2/3 antibody (D7G7 XP rabbit mAb #8685, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. For detection, the Alexa Fluor 488-conjugated antibody (Cell Signaling Technology) was used. Images were captured with a fluorescent microscope (Axio Imager M2, Carl Zeiss AG, Oberkochen, Germany).
6
Cell Cycle and Apoptosis Analysis
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Cell cycle distribution including the pre-G1 fraction was determined from DNA histograms as described before [54 (link)]. Apoptosis was quantified from detection of active, cleaved caspase-3 by flow cytometry using an Alexa Fluor® 488-conjugated antibody (Cell Signaling Technology, Danvers, Massachusetts, USA) according to the manufacturer's protocol. Cell proliferation and the rate of DNA synthesis were determined by flow cytometry on AGS cells labelled with anti-BrdU antibody (AbD Serotec, Puchheim, Germany) and propidium iodide (PI, Sigma-Aldrich) according to the manufacturer's protocol. Briefly, cells were pulse labelled with 10 μM BrdU (Sigma-Aldrich) for 60 min, acid-treated and stained with an anti-BrdU and a secondary APC-conjugated antibody for determination of DNA synthesis and with 20 μg/ml PI for determination of total DNA content.
7
Immunostaining of α-SMA in Tissue Slices
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The slices seeded with PAFs were fixed in paraformaldehyde for 30 min at room temperature. 0.5%Triton-X was used to penetrate the cells. To prevent nonspecific background staining, the slices were blocked by 5% BSA for 1 h. Then slices were incubated with primary antibody α-SMA (1:100, Proteintech, 14395-1-AP) overnight at 4 °C, followed by incubation with corresponding secondary antibody (1:1000, goat-anti-rabbit Alexa Fluor 488-conjugated antibody, Cell signaling technology) for 1 h at room temperature. The nuclei were stained with DAPI (1:2000, Sigma-Aldrich, USA) for 30 min at room temperature. The fluorescence was captured with a fluorescence microscope (Leica TCS-SP5II, Germany). Three images of each simple were used for quantification.
8
Immunofluorescence Analysis of p65 in HeLa Cells
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HeLa cells were grown overnight to 70% confluence in 24-well plates and infected for the indicated time. Cells were then washed, fixed and stained with anti-p65 (SC-372, Santa Cruz), phalloidin-rhodamine (P1951, Sigma) and DAPI (D9542, Sigma), followed by washing and staining with anti-rabbit Alexa Fluor 488 conjugated antibody (#4812, Cell Signaling). Cells were visualized using fluorescence microscopy. All experiments were repeated at least three times and significance was tested using the student T-test (un-paired, two-tailed).
9
Immunofluorescence Analysis of Transfected HEK293T Cells
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HEK293T cells were grown in round glass coverslips and post transfection with the indicated plasmids, the cells were fixed and permeabilized with ice-cold methanol and then washed. The non-specific binding sites were blocked with BSA 1% PBS for 30 min. Then, cells were incubated ON at 4°C with anti-FLAG. Secondary Alexa Fluor 488-conjugated antibody (#4414, Cell Signaling Technologies) was incubated for 1h at room temperature. Negative controls were carried out using PBS 1% BSA instead of the primary antibody. Nuclei were stained with DAPI 2 µg/ml (Sigma Aldrich, Saint Louis, MO). Finally, the coverslips were mounted on glass slides with mounting medium (Prolong Gold Antifade Reagent, ThermoFisher). Cells were analyzed using an Olympus DSU IX83 Spinning Disk microscope.
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