The sections were blocked by incubation with 4.5% normal goat serum (NGS) in PBS for one hour, then incubated overnight at 4 °C in PBS supplemented with 3% NGS, 0.2% Triton X-100 and one of the following antibodies: anti-alpha-synuclein (1:500; mouse; (211) Santa-Cruz), anti-phospho S129-alpha-synuclein (1:500; rabbit; EP1536Y, Abcam), anti-TH (1:1000; rabbit; Millipore), anti-HSC70 (1:500; mouse; B-6, Santa-Cruz), anti-LAMP2A (1:500; rabbit; EPR4207(2), Abcam), anti-GFAP (1:500; rabbit; Millipore), anti-S100β (1:100; rabbit; EP1576Y, Abcam), anti Iba1 (1:1000; rabbit; EPR16588), Abcam), anti-acetylated lysines (1:100; rabbit; Millipore). Sections were rinsed three times in PBS and incubated with Alexa-Fluor 488-labeled anti-mouse IgG or Alexa Fluor 594-labeled anti-rabbit IgG (Life Technology) for 1 h at room temperature. The sections were rinsed in PBS and the nuclei were counterstained with DAPI (dilution 1:10,000, Wako) for 3 minutes. The sections were mounted on glass slides and coverslips in Aqua-Poly/Mount (Polysciences), and stored at 4 °C in the dark until further processing. Images of the immunostained sections were taken within 48 h.
Alexa fluor 594 labeled anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-labeled anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 labeled anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Epr4207 2, by Abcam (1 mentions)
Ep1536y, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Fujifilm (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Bs 1164r, by Bioss Antibodies (1 mentions)
Lectin, by Vector Laboratories (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Parkin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Collagen type 3, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 labeled anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Mnsod, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Microfil, by Flow Tech (1 mentions)
Mouse anti α tubulin, by Cell Signaling Technology (1 mentions)
Rabbit anti crbn antibody, by Merck Group (1 mentions)
Mg132, by Peptide Institute (1 mentions)
6 protocols using alexa fluor 594 labeled anti rabbit igg
1
Immunohistochemical Analysis of Alpha-Synuclein and Related Markers
2
Comprehensive Histological Analysis of Metabolic Tissues
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Liver, gastrocnemius muscle, and sWAT samples were fixed with 4% formaldehyde in phosphate buffer (Histolab Products, Gothenburg, Sweden), embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H-E). Liver samples were also embedded in optimal cutting temperature mounting medium (Histolab Products) and frozen in isopentane cooled by dry ice followed by cryosectioning and Oil Red O staining. Total hepatic lipid area and lipid droplet size distribution and average muscle fiber diameter and fiber size distribution were assessed in five randomly selected 20× microscopic fields per mouse using ImageJ version 1.47 software (National Institutes of Health, Bethesda, MD). For immunofluorescence, gastrocnemius muscle sections were incubated with rabbit anti-perilipin 2 antibody (bs-1164R; Bioss, Woburn, MA) followed by incubation with Alexa Fluor 594–labeled anti-rabbit IgG (A21207; Life Technologies, Grand Island, NY). Quantification of immunofluorescence was based on three randomly selected 10× microscopic fields per mouse.
3
Vasculogenesis and Mitochondrial Dynamics
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Ang II and β‐actin antibodies were purchased from Sigma Chemicals. Microfil was obtained from Flow Tech, Inc. Collagen type I, collagen type III, aldehyde dehydrogenase 2, vascular endothelial growth factor (VEGF), Pink, and Parkin antibodies were obtained from Santa Cruz Biotechnology. MnSOD and anti‐4‐hydroxy‐nonenal antibodies were purchased from Millipore and Calbiochem, respectively. Antibodies against SIRT3, LC3B, and α‐smooth muscle actin were obtained from Cell Signaling Technology. Secondary antibodies, including peroxidase‐conjugated rabbit anti‐goat IgG, rabbit anti‐mouse IgG, and goat anti‐rabbit IgG, were supplied by Life Technologies. Matrigel and collagen I rat tail protein were obtained from Corning. Lectin was purchased from Vector Laboratories. SIRT3‐short hairpin RNA (shRNA) and SIRT3‐overexpressing lentivirus were synthesized by Genechem Corporation. Alexa Fluor 555‐labeled anti‐mouse IgG, Alexa Fluor 594‐labeled anti‐rabbit IgG, Alexa Fluor 488‐labeled anti‐rabbit IgG, MitoTracker Red, MitoTracker Green, and wheat germ agglutinin were from Life Technologies. Cell culture reagents were purchased from Hyclone.
4
Immunoblotting and Immunofluorescence Techniques
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The following antibodies were used: mouse anti-hemagglutinin (HA) antibody (Covance, Berkley, CA, USA); mouse anti-myc antibody (Millipore, Billerica, MA, USA); rabbit anti-CRBN antibody (Sigma Aldrich, St. Louis, MO, USA); mouse anti-α-tubulin (Cell Signaling Technologies, Beverly, MA, USA); rabbit anti-Tom20 (F-10) (Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-DLP1 antibody (BD Bioscience, San Jose, CA, USA); mouse anti-OPA1 antibody (BD Bioscience); anti-rabbit IgG, HRP-linked whole antibody, donkey (GE Healthcare, Waukesha, WI, USA); anti-mouse IgG, HRP-linked whole antibody, sheep (GE Healthcare); Alexa Fluor488-labeled anti-mouse IgG (Life Technologies, Carlsbad, CA, USA); and Alexa Fluor594-labeled anti-rabbit IgG (Life Technologies); rabbit polyclonal anti-dinitrophenyl (DNP) antibody (SHIMA Laboratories, Tokyo, Japan). MG132 (Peptide Institute, Osaka, Japan) was dissolved in dimethyl sulfoxide and stored at −20 °C.
5
Quantifying Nav1.7 Subcellular Distribution
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Transfected HEK293 cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton™ X-100 for 15 min, and blocked with 5% BSA for 30 min at room temperature. Cells were incubated with Nav1.7 polyclonal antibodies (1:200) overnight at 4°C and then incubated with secondary Alexa Fluor 594 labeled anti rabbit IgG (1:500, Life Technologies) for 90 min at 37°C. After that, cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Images were captured at ×60 magnification using an OLYMPUS FV1000 confocal microscope system. To quantify the subcellular distribution of Nav1.7, fluorescence intensity at the entire cell area was measured with the ROI Manager using ImageJ software, and at least 100 transfected cells were analyzed for each construct.
6
Immunostaining and PTH Administration for Bone Analysis
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For immunostaining of histological sections, mice were perfused with 4% paraformaldehyde followed by PBS. Decalcified tibiae were embedded in paraffin and the sections were prepared. Apoptotic cells were detected using the ApopTag Peroxidase in Situ Apoptosis Detection Kit (Merck Millipore). Methyl green was used for nuclear counterstaining. For immunofluorescent staining, mice were perfused with 4% paraformaldehyde followed by PBS. Femur were further fixed with 4% PFA for 1hr at 4 C. They were equilibrated with 30% sucrose in PBS at 4 C over night and embedded in SCEM (Leica Microsystems). Frozen sections were prepared as described previously (Kawamoto and Kawamoto, 2014) . The sections were subjected to immunostaining with anti-osteocalcin and anti-GFP antibodies to identify osteoblasts and IL-7-GFP, respectively. Antibodies used in this study were: anti-osteocalcin (Takara Bio Inc.), anti-GFP (catalog no. A10262, Life technologies), Alexa Fluor 488labeled anti-chicken IgY (A11039) and Alexa Fluor 594-labeled anti-rabbit IgG (A11012) antibodies.
In Vivo PTH Administration Human PTH (1-34) (Bachem) (40 mg per kg body weight) or saline was administered intraperitoneally once daily for 2 weeks before cecal ligation and puncture.
In Vivo PTH Administration Human PTH (1-34) (Bachem) (40 mg per kg body weight) or saline was administered intraperitoneally once daily for 2 weeks before cecal ligation and puncture.
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