In order to perform immunofluorescence staining for muscle sections, we collected mouse GAS specimens, froze them with liquid nitrogen in tissue-Tek OCT, and then sliced the muscle sections into 20um with Cryostat (CM1950, Leica). The muscle sections were fixed with paraformaldehyde PFA (4%, 15 min), washed by PBS 3 times, infiltrated by Triton x-100 (0.1%, 10 min) and blocked by Mouse on Mouse (M.O.M.). Block reagent (carrier laboratory) and 5% BSA/5% normal goat serum /PBS were incubated with primary antibody. Antibodies used included mouse anti-MyHC I (BA-D5-S 1:100, DSHB), mouse anti-MyHC IIb (BF-F3 1:100, DSHB) and rabbit anti-laminin (AB2034 1:500, Millipore). Sections were rinsed with PBS/0.1% Tween-20 and incubated with Alexa Fluor 488 or Alexa Fluor 568 labeled secondary antibody (Invitrogen, 1:800). Slides were imaged on LEICA TCS SP8 (LEICA, Germany) confocal microscope.
Alexa fluor 568 labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568-labeled secondary antibody is a fluorescent-conjugated antibody that binds to the primary antibody. It is used in various immunodetection techniques, such as immunofluorescence and Western blotting, to visualize and localize specific target proteins.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 labeled, by Thermo Fisher Scientific (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Dapi fluoromount g solution, by Electron Microscopy Sciences (2 mentions)
Rabbit anti a fumigatus antibody, by Meridian Bioscience (2 mentions)
Tcs sp8, by Leica (1 mentions)
Cryostat, by Leica (1 mentions)
Ab2034, by Merck Group (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
μ slide 8 well, by Ibidi (1 mentions)
Lsm700 axioobserver, by Zeiss (1 mentions)
Ab20194, by Abcam (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Anti flag antibody, by Abcam (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
12 protocols using alexa fluor 568 labeled secondary antibody
1
Immunofluorescence Staining of Mouse Muscle
2
Immunofluorescence Imaging of Transfected Cells
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BHK cells were seeded on ibiTreated 8-well μ-slides (Ibidi) and transfected with the indicated plasmids. After 16 h of incubation at 37 °C, the cells were fixed with cold methanol and incubated with either anti-HSV-1 ICP8 (ab20194, Abcam), anti-HA (H9658), or anti-FLAG (F3165) followed by incubation with Alexa Fluor 568-labeled secondary antibody (A11004, Invitrogen). Finally, the cell nucleus was stained with DAPI (Invitrogen). Confocal images were acquired using a Carl Zeiss LSM 700 Axio Observer. Z1 inverted the confocal laser-scanning microscope with either a plan-apochromat 20×/0.8 objective or with a plan-apochromat 63×/1.4 oil objective. Merging of images and profile plot measurements were done with the program ImageJ. Images were further processed for publication with Adobe Illustrator CS6.
3
Immunofluorescence Analysis of ETS1 Protein
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The cells were cultured on glass coverslips (Blue Star 22 × 22 mm square No. 1 cover glass sterilized with 70% methanol) and were given treatment after serum starvation. After discarding the medium, the cells were fixed with 4% paraformaldehyde (pH 7.4) for 20 min followed by permeabilization with 0.1% Triton X-100 for 15 min at room temperature. Then the fixed cells were incubated overnight with anti-ETS1 antibody (1:200; Cell Signaling Technology Cat# 6258BC, Lot # 1) in 4 °C after 1 h of blocking in 3% BSA in PBS (HIMEDIA, India, Cat# TL1101) solution. Then the cells were stained with either Alexa-Fluor 488-labeled secondary antibody (1:400; Invitrogen, Cat# A11008, Lot# 2051237) or Alexa-Fluor 568-labeled secondary antibody (1:400; Invitrogen, Cat# A-21069, Lot# 108193) for 2 h in room temperature and the nucleus was stained with DAPI (0.25 μg/ml) for 5 min. To stain the filamentous actin, after permeabilization cells were incubated with Rhodamine-phalloidin (Cat# R415, Invitrogen) for 20 min in room temperature. Images were taken using TCS SP8 confocal microscope (Leica Microsystems, RRID: SCR_008960) with 63X oil immersion objective lens and Olympus BX51 microscope (Olympus, RRID: SCR_017564) with 40X objective lens.
4
Subcellular Localization of LOX in HEK293 Cells
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HEK293 cells were maintained in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37℃ in 5% CO 2 . For transfection, 8.0 × 10 5 HEK293 cells were plated in gelatin coated cover slips in 6 well plates, grown for 18 h, and then transfected with a plasmid encoding LOX-FLAG or LOX-v2-FLAG, using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. After 6 h of transfection, the medium containing the plasmid DNA complexes was replaced by the fresh medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. At post-transfection 48 h, cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. The cells were blocked with 3% goat serum for 30 min, washed with PBS, and then incubated anti-FLAG antibody (Abcam, Cambridge, MA, USA; 1:250). Staining was revealed by an Alexa Fluor 568-labeled secondary antibody (Invitrogen, 1:1,000). Nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA; 1:1,000). Confocal images were scanned using the Fluoview FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
5
Immunofluorescence Staining of Ki67
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Cells were fixed using ice-cold acetone (Sigma Aldrich, UK) for 15 min. Following fixation, cells were rinsed with PBS and subsequently incubated with blocking solution for 30 min and then with primary Ki67 antibody (1:200; Cat. No. 9129, Cell Signaling Technology, USA) overnight in a cold room. The next day, cells were rinsed and incubated with Alexa-Fluor 568-labeled secondary antibody (1:1000; Molecular Probes, USA) for 1 h in the dark. The secondary antibody alone was used as a negative control. Cell nuclei were stained using DAPI reagent (Vector Laboratories, UK). Subsequently, cells were rinsed and mounted onto glass slides using Mowiol reagent with 10% Mowiol D488 (Calbiochem, UK) and stored in a freezer. Staining was visualized with a Leica DMLB microscope. Cell images were acquired using a CCD camera (Cool-SNAP-Pro, Media Cybernetics, USA) with Image-Pro Plus software version 6.0 (Media Cybernetics, USA).
6
Double-Fluorescence Staining of Cellular Markers
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Double-fluorescence staining was performed as previously described [26, 30] . Briefly, deparaffinized sections were incubated in citrate buffer solution (pH 6.0), and their antigenicities were enhanced in an autoclave (121°C, 1 min). After treatment to block endogenous peroxidase activity and non-specific binding, the sections were incubated with biotinylated lectins diluted in 1% BSA in PBS for 40 min. After rinsing the sections with PBS, they were incubated with fluorescein isothiocyanate (FITC)-labeled streptavidin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. The sections were then incubated overnight at 4°C with a primary rabbit anti-GM130 monoclonal antibody (Abcam, Cambridge, UK) or a primary rabbit anti-K5 polyclonal antibody (Covance, Berkeley, CA, USA). After washing with PBS, sections were stained for 30 min with an Alexa Fluor ® 568-labeled secondary antibody (Molecular Probes, Eugene, OR, USA). Sections were counterstained with Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA), and imaged using a BX-51 fluorescence microscope (Olympus, Japan). Control samples were prepared by omitting the primary antibody incubation step.
7
Immunofluorescence Staining of Myogenic Markers
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The obtained cells were fixed with 4% parafor-maldehyde for 10 min at room temperature, washed, and treated with 0.1% Triton X-100/phosphate-buffered saline (PBS) for 10 min for permeabilization. The cells were blocked with 5% goat serum (Cedarlane) in 2% bovine serum albumin (BSA)/PBS for 15 min, and then incubated with anti-Pax7 (1:80, sc-81648, Santa Cruz), anti-MyoD (1:400, sc-32758, Santa Cruz) or anti-Myogenin antibody (1:1000, sc-12732, Santa Cruz) in 2% BSA/PBS at 4°C overnight. The cells were then incubated with Alexa Fluor 488- or Alexa Fluor 568-labeled secondary antibodies (1:1000, Thermo Fisher Scientific) in 2% BSA/PBS. After several washings, nuclei were stained with Hoechst (DOJINDO). Immunofluorescent images were evaluated by fluorescence microscopy (Olympus).
8
Fluorescent Protein Localization in Cells
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For cell staining, A375 cells expressing shTIM (shMock, sh1, sh2) and shTIPIN (shMock, sh1, sh2) were incubated with the primary antibody. One day later, Alexa Fluor 488-labeled or Alexa Fluor 568-labeled secondary antibodies (Thermo Scientific) were added. The DAPI-Fluoromount-G® solution (Electron Microscopy Sciences, Hatfield, PA, USA) was added for nuclei staining of the cells. Fluorescence-labeled proteins were analyzed using the Zeiss fluorescent microscope with the apotome attachment and Zeiss software and Axiocam cameras (Carl Zeiss Microscopy GmbH, Deutschland).
9
Mitochondrial Staining of Colon Cancer Cells
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For mitochondrial staining, HCT-116p53+/+ and HCT-116p53−/− live cells were first incubated with 0.3 μM MitoTracker Red CMXRos (Invitrogen, Eugene, Oregon, USA) for 30 min at 37 °C in 5% CO2. For co-localization and for mitochondrial staining, colon cancer cell lines were fixed with 3.7% formaldehyde, and then permeabilized in 300 μl of 0.5% Triton X-100. In general for immunostaining, after blocking the cover slips in 10% BSA for 1 h, the primary antibody was added overnight. The day after, Alexa Fluor 488-labeled or Alexa Fluor 568-labeled secondary antibodies (Thermo Scientific) were added (see Table S2 ). The DAPI-Fluoromount-G® solution (Electron Microscopy Sciences, Hatfield, PA, USA) was added for nuclei staining of the cells. Fluorescence-labeled proteins were analyzed using the Zeiss Fluorescent microscopy with apotome attachment and the Zeiss software and Axiocam cameras (Carl Zeiss Microscopy GmbH, Deutschland).
10
Endocytosis of A. fumigatus by Host Cells
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