Animals were deeply anesthetized by pentobarbital (Somnopentyl, Kyoritsu Seiyaku Company Limited, Tokyo, Japan) and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were post-fixed with 4% paraformaldehyde in PBS and cryopreserved overnight in 2% paraformaldehyde/15% sucrose in PBS and then maintained in 30% sucrose in PBS. The brains were sectioned on a freezing microtome (REM-710, Yamato, Saitama, Japan) at 50 μm thickness. For enhancement of native signals, some brain sections were processed for immunostaining as described previously (Osakada et al., 2008 (link), 2011 (link)). The primary antibodies and their working dilutions were as follows: rabbit anti-GFP polyclonal (1:2,000, Abcam, UK) and rabbit anti-DsRed polyclonal (1:1,000, Clontech-Takara, Japan). Labeled cells were visualized with the following fluorescent secondary antibodies: anti-mouse IgG and anti-rabbit IgG conjugated with Alexa Flour 488 or Alexa Flour 594 (1:1,000, Jackson Immunoresearch, West Grove, PA, USA). The sections were mounted on slide glasses with anti-fade solution (Nakalai, Kyoto, Japan). Labeled cells were imaged with a confocal laser-scanning microscope with GaAsP detectors (LSM800, Zeiss, Jena, Germany) using a 10× (NA 0.45, Zeiss, Jena, Germany), 20× (NA 0.75, Zeiss, Jena, Germany), or 40× objective lens (NA 1.2, Zeiss, Jena, Germany).
Alexa flour 594
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 594 is a fluorescent dye manufactured by Jackson ImmunoResearch. It is an amine-reactive dye with an excitation maximum at 588 nm and an emission maximum at 618 nm. Alexa Fluor 594 can be used for various fluorescence-based applications, including flow cytometry, immunofluorescence, and microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Dab substrate, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Hematoxylin, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Pnfkb, by Cell Signaling Technology (2 mentions)
Ptgs2 160112, by Cayman Chemical (2 mentions)
T6079, by Merck Group (2 mentions)
Total nf kb, by Cell Signaling Technology (2 mentions)
Lsm 800, by Zeiss (1 mentions)
Rabbit polyclonal anti gfp, by Abcam (1 mentions)
Rem 710, by Yamato Scientific (1 mentions)
Bz 2 analyzer software, by Keyence (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Cyanine cy3, by Jackson ImmunoResearch (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
6 protocols using alexa flour 594
1
Immunostaining and Confocal Imaging of Brain Tissue
2
Immunostaining of Muscle Tissue and Cells
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Hind limb muscles were excised, fixed in 4% PFA, cryoprotected with 30% sucrose, and embedded in Tissue-Tek optimum cutting temperature formulation. Sections were cut transversely and subjected to immunostaining and nuclei counterstain with DAPI. C2C12 myoblasts were fixed in 4% PFA and subjected to immunostaining and nuclei counterstain with DAPI. Fixed tissues and cells were permeabilized with 0.05% Triton X-100 for 20 min and blocked with 5% normal donkey serum. Primary antibodies were visualized with cyanine Cy3 or Alexa-Flour 594 conjugated donkey anti–mouse antibodies from Jackson ImmunoResearch Laboratories. Coverslips were mounted with Aqua-Poly/Mount (Polysciences) or Vectashield (Vector Laboratories). All images were acquired at RT with a BZ-9000 fluorescence microscope (KEYENCE) using either a 10× PlanApo (NA 0.45) or a 20× PlanApo (NA 0.75) objective with the BZ-II Analyzer software (KEYENCE). Approximately 10–15 images were acquired from each transverse section. Myofiber cross-sectional areas were calculated using BZ-II Analyzer software or CellProfiler (Jones et al., 2008 (link)).
3
Immunohistochemical and Immunocytochemical Analyses
4
MBP Protein Detection in BD Patients
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The standard reaction mixture after incubation with IgG of BD patients or healthy individuals was separated on SDS-PAGE, as described above in Section 4.4 and transferred to polyvinylidene difluoride (PVDF) membranes using electroblotting in tris glycine transfer buffer. The blot was pre-incubated for 1 h in a blocking buffer (5% BSA in phosphate-buffered saline (PBS)), then in primary anti-myelin basic protein (MBP) antibodies overnight at 4 °C at concentrations of 1:2000 (Aves Labs, Davis, CA, USA). The blot was washed with PBS containing 0.05% Tween (Sigma Aldrich, Saint Louis, MO, USA) (3 × 10 min), and incubated for 1 h with the secondary antibody conjugated with Alexa Flour 594 (1:2000, Jackson ImmunoResearch, West Grove, PA, USA). The blot was washed again in PBS-Tween (3 × 10 min) and the bands were visualized using the iBright Imaging Systems FL1500 (Thermo Scientific, Waltham, MA, USA).
5
Immunohistochemical Analysis of Ki-67 and CD31
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Following the deparaffinization of the sections in xylene, they were placed at room temperature for an overnight incubation with primary antibodies against Ki-67 and CD31 (1 : 500, Abcam, Cambridge, UK). Goat secondary antibodies conjugated with Alexa Fluor 488 and Alexa Flour 594 (Jackson ImmunoResearch, West Grove, PA, USA) were added, and the samples were incubated for 1 h at room temperature. Nuclei were stained with DAPI, and fluorescent images were obtained using a fluorescence microscope. All experiments were performed in triplicate.
6
Immunohistochemical and Immunocytochemical Analyses
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Paraffin-embedded slides were deparaffinized, dehydrated, and rehydrated using a series of acetone and alcohol washes. Antigen retrieval was performed using citrate buffer at pH 6.0 in a pressure cooker. The primary antibody (PTGS2: 160112, 1:100; Cayman Chemicals) was prepared in 4% fish gelatin and incubated with the slides overnight at 4°C. Slides were then washed and incubated with HRP-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA) at room temperature for 1 hour. DAB substrate (Invitrogen, Grand Island, NY) was used to visualize staining of protein and hematoxylin (Invitrogen) was used to stain nuclei. For immunocytochemistry experiments, Skov3-ip1 and HeyA8 cells were grown in chamber slides prior to treatment with NE. Cells were fixed in 4% paraformaldehyde and permeabilized using Triton X-100. Staining using primary (PTGES: 1:100; T6079, Sigma Aldrich, p-Nf-kB: 1:100; 3033S, Cell Signaling, total Nf-kB: 1:100, 8242S, Cell Signaling) and secondary antibodies (anti-rabbit Alexa Flour 594, 1:800; Jackson Immuno Research) was done as described above. Hoechst (Invitrogen) was used to stain nuclei.
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