Immunodetection of neuronal markers was carried out as previously described [53 (link), 54 (link)]. Primary antibodies used were: rabbit anti-doublecortin (Cell Signaling Technology Inc.), monoclonal anti-NeuN (Millipore), goat anti-FZD1 (R&D Systems), goat anti-FZD1 (LifeSpan Biosciences, Inc.), rabbit anti-SOX2 (Cell Signaling Technology Inc.), monoclonal anti-GFAP (Sigma-Aldrich), monoclonal anti-Nestin (Millipore), rabbit anti-Ki67 (Abcam). As secondary antibodies, Alexa (Molecular Probes) and DyLight (Abcam) conjugated antibodies were used. NucBlue (Life Technologies) was used as nuclear dye. Slices were mounted on gelatin-coated slides with Fluoromont-G (Electron Microscopy Sciences). Double-labeled sections were analyzed by confocal laser microscopy (Olympus FV 1000). Image analysis and z-projections were made with ImageJ software (NIH, USA).
Fluoromont g
Manufactured by Electron Microscopy Sciences
Sourced in United States
Fluoromont-G is a fluorinated mounting medium for use in electron microscopy. It is designed to provide a stable and inert environment for the preservation and analysis of biological specimens during electron microscope examination.
Automatically generated - may contain errors
Lab products found in correlation
Nucblue, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ki67, by Abcam (2 mentions)
Rabbit anti sox2, by Cell Signaling Technology (1 mentions)
Rabbit anti doublecortin, by Cell Signaling Technology (1 mentions)
Monoclonal anti neun, by Merck Group (1 mentions)
Monoclonal anti gfap, by Merck Group (1 mentions)
Monoclonal anti nestin, by Merck Group (1 mentions)
Goat anti fzd1, by R&D Systems (1 mentions)
Fv1000, by Olympus (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Goat anti dcx, by Santa Cruz Biotechnology (1 mentions)
Goat anti sox2, by R&D Systems (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti dcx, by Cell Signaling Technology (1 mentions)
Rat anti brdu, by Abcam (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
4 protocols using fluoromont g
1
Immunodetection of Neuronal Markers
2
Immunohistochemical Analysis of Leptin Receptor Activation
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Floating sections were rinsed with PBS and blocked with 3% normal donkey serum in PBS + 0.25% Triton-X 100 for 1 hour at room temperature. Sections were incubated overnight at 4°C with primary antibodies in blocking buffer. Primary antibodies used are listed in Table 2 . Sections were rinsed and incubated with secondary antibodies for 1 hour and rinsed in PBS. Secondary antibodies used are listed in Table 2 . Sections were mounted on gelatin-precoated slides and coverslipped with Fluoromont-G (Electron Microscopy Sciences) mounting medium.
pSTAT3 immunoreactivity was performed to assess reactivation of functional leptin receptor. Brain sections were pretreated with 30% H2O2 to block endogenous peroxidase activity, 0.3% glycine and 0.03% sodium dodecyl sulphate (SDS) prior to primary antibody incubation. Tissue was blocked in 3% normal donkey serum + 0.25% Triton in PBS for 1 hour and incubated in primary rabbit anti-pSTAT3 (Table 2 ) for 48 hours at 4°C. Primary antibody was detected using a biotinylated secondary antibody (Table 2 ) by avidin-biotin peroxidase method (ABC Kit, Vector Laboratories) using diaminobenzidine (DAB; Sigma) as chromogen.
pSTAT3 immunoreactivity was performed to assess reactivation of functional leptin receptor. Brain sections were pretreated with 30% H2O2 to block endogenous peroxidase activity, 0.3% glycine and 0.03% sodium dodecyl sulphate (SDS) prior to primary antibody incubation. Tissue was blocked in 3% normal donkey serum + 0.25% Triton in PBS for 1 hour and incubated in primary rabbit anti-pSTAT3 (
3
Immunostaining of Neurogenesis Markers
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Immunodetection of proliferation, progenitor and neuronal markers, and BrdU was carried out as previously described7 (link),93 (link). Primary antibodies used were rabbit anti-DCX (1:750, Cell Signaling Technology Inc.), goat anti-DCX (1:250, Santa Cruz Biotechnology), rabbit anti-Ki67 (1:250, Abcam), mouse anti-NeuN (1:300, Millipore), mouse anti-GFAP (1:8000, Sigma), goat anti-Sox2 (1:2000, R&D Systems), rat anti-BrdU (1:300, Abcam). As secondary antibodies Alexa (1:500, Molecular Probes) and DyLight (1:500, Abcam) conjugated antibodies were used. NucBlue (Life Technologies) was used as nuclear dye. Slices were mounted on gelatin-coated slides with Fluoromont-G (Electron Microscopy Sciences).
4
Culturing and Characterizing HeLa Cell Lines
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HeLa cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum supplemented with 50 U/ml of penicillin and streptomycin, at pH 7.4 and kept at 37°C in 5% CO2/95% air atmosphere. HeLa-Cx39 and HeLa Cx43-EGFP transfected cells were selected with 0.5 mg/mL of puromycin and 0.3 mg of geneticin (Invitrogen, MA, USA), respectively, as described previously (Jordan et al., 1999 (link); von Maltzahn et al., 2004 (link)). The Hela-Cx39 cells were kindly provided by Dr. Klaus Willecke, Lymes Institute, University of Bonn, Bonn, Germany. For each experiment, cells were seeded on glass coverslips, until appropriate confluence was reached. Cells were always fed the day before an experiment.
The Cx39 transfection was confirmed by immunofluorescence. In brief, cells on glass coverslips, were washed with PBS, and fixed with formaldehyde 4% and incubated with blocking solution (1% BSA, 0.025% triton X-100 and 50 mM NH4Cl, in PBS, at pH 7.4) overnight. Then, cells were incubated with rabbit anti-Cx39 antibody (Invitrogen, MA, USA) for 6 h at room temperature, washed with PBS and incubated 30 min with secondary rabbit anti-IgGs (Santa Cruz, Texas, USA) conjugated to Cy2 or Cy3. Preparations were subsequently washed with PBS, and coverslips were mounted with fluoromont G (Electron Microscopy Science, PA, USA).
The Cx39 transfection was confirmed by immunofluorescence. In brief, cells on glass coverslips, were washed with PBS, and fixed with formaldehyde 4% and incubated with blocking solution (1% BSA, 0.025% triton X-100 and 50 mM NH4Cl, in PBS, at pH 7.4) overnight. Then, cells were incubated with rabbit anti-Cx39 antibody (Invitrogen, MA, USA) for 6 h at room temperature, washed with PBS and incubated 30 min with secondary rabbit anti-IgGs (Santa Cruz, Texas, USA) conjugated to Cy2 or Cy3. Preparations were subsequently washed with PBS, and coverslips were mounted with fluoromont G (Electron Microscopy Science, PA, USA).
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