For the histological analysis of aortic tissue, aortas were removed from PBS-perfused mice, fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 6 μm thickness. Slides were stained with hematoxylin and eosin (H&E; BBC, Biochemical), Masson’s trichrome (MT; Abcam, ab150686), and Verhoeff-Van Gieson (VVG). For the immunohistochemical examination, deparaffinized samples were boiled in Tris-EDTA buffer (pH 9.0, 0.05% Tween-20) for antigen retrieval, followed by a standard protocol for immunostaining. For analysis of cell death, aortic sections were processed by using a Click-iT TUNEL assay imaging kit (Invitrogen, C10243). Immunocytochemistry was performed as previously described36 (link). In brief, cells cultured on coverslips were fixed using 4% PFA and permeabilized with 0.1% Triton X-100 in PBS. Cells were then blocked with 10% goat serum diluted in PBS. The cells were incubated with primary antibodies diluted in the blocking solution overnight at 4 °C. The cells were incubated with Alexa 488- or Alexa 568-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature (RT). The stained cells were mounted with Mowiol solution. Fluorescent images were obtained using an LSM-710 confocal microscope system (Carl Zeiss) and ZEN software (Carl Zeiss).
Alexa 488 or alexa 568 conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies are fluorescently-labeled antibodies that can be used to detect and visualize target proteins in various applications, such as immunofluorescence microscopy and flow cytometry. These antibodies are designed to specifically bind and recognize primary antibodies that have been raised against a target protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (4 mentions)
Mounting medium, by Vector Laboratories (3 mentions)
Lsm 710 confocal microscope system, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Ab167453, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Nb600 308, by Novus Biologicals (1 mentions)
Imager a1 fluorescence microscope, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Bz 9000, by Keyence (1 mentions)
Bz 2 analyzer software, by Keyence (1 mentions)
Carl lsm 710 confocal microscope, by Zeiss (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
7 protocols using alexa 488 or alexa 568 conjugated secondary antibody
1
Histological and Immunohistochemical Analysis of Aortic Tissue
2
Spinal Cord Tissue Immunofluorescence
3
Immunofluorescence Analysis of Lung Tissue
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Fresh lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. After deparaffinating, rehydration and retrieval, tissues were incubated with primary antibodies overnight at 4°C. After rinsing several times with PBS, sections were incubated with Alexa-488- or Alexa-568-conjugated secondary antibodies (Invitrogen, United Kingdom) for 2 hours. Finally, DAPI was added onto slides for 10 minutes. Images were obtained with a ZEISS Imager A1 fluorescence microscope (Gottingen, Germany). Immunofluorescence was performed using the following primary antibodies: rabbit monoclonal anti-P21 and anti-PTEN antibodies (Abcam, United Kingdom); and mouse monoclonal anti-SPC antibody (Santa Cruz Biotechnology, CA, USA).
4
Immunofluorescence Imaging of Vascular Smooth Muscle Cells
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VSMCs cultured on coverslips were fixed using 4% paraformaldehyde and then incubated with a blocking solution (0.1% Triton X-100, 0.2% BSA, and 10% normal goat serum, in PBS [Nacalai Tesque]). The cells were incubated with the primary antibodies diluted in the Can Get Signal immunostain reagent (TOYOBO) and then incubated with Alexa 488– or Alexa 568–conjugated secondary antibodies (Invitrogen) diluted in the blocking solution. To label the nuclei, Hoechst 33342 (Invitrogen) was added to the secondary antibody solution. The stained cells were mounted with Fluoromount (Diagnostic BioSystems) and observed using an all-in-one fluorescence microscope (BZ-9000; Keyence) with a CFI Plan Apochromat λ 20× lens (Nikon) at room temperature. Fluorescent images were acquired with an internal charge coupled device camera using BZ-II Analyzer software (Keyence) and processed with Photoshop Element 10 software (Adobe).
5
Immunostaining of Drug-Treated Cells
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Cells were grown on sterilized glass coverslips. After drug treatment, the cells were fixed with 4% paraformaldehyde. Thereafter, the cells were blocked with 10% goat serum in PBS, stained with a 1:500 dilution of primary antibody in PBS, and then reacted with a 1:1,000 dilution of Alexa 488- or Alexa 568-conjugated secondary antibody (Invitrogen) for immunostaining. Finally, the slides were washed three times with PBS, stained with DAPI, and mounted in mounting medium (Vector, USA). Images were captured with a Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Germany).
6
Immunostaining and Confocal Microscopy
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Cells were grown on sterilized glass coverslips. After drug treatment, the cells were fixed with 4% paraformaldehyde. For immunostaining, cells were blocked with 10% bovine serum albumin in PBS and stained with a 1:1,000 dilution of primary antibody in PBS, and then reacted with a 1:1,000 dilution of Alexa 488- or Alexa 568-conjugated secondary antibody (Invitrogen). Finally, the slides were washed three times with PBS, stained with DAPI, and mounted in mounting medium (Vector, Burlingame, CA, USA). Images were captured with a Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochem, Germany). GFP-mRFP-LC3 (ptfLC3) constructs were purchased from Addgene (Cambridge, MA, USA).
7
Immunostaining and Confocal Imaging
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Cells were grown on sterilized glass coverslips and fixed with 4% paraformaldehyde after treatment with 6-AZA. For immunostaining, cells were blocked with 10% goat serum in phosphate-buffered saline (PBS), stained with a 1:500 dilution of primary antibody in PBS, and then reacted with a 1:1000 dilution of Alexa 488- or Alexa 568-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Finally, the slides were washed three times with PBS, stained with DAPI, and mounted on glass slides using a mounting medium (Vector, Burlingame, CA, USA). Images were acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). LysoTracker was purchased from Invitrogen, and the antibody for LAMP1 was purchased from Santa Cruz Biotechnology.
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