In all, 10- to 12-week-old mice (n = 7–8 per group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-µm thick, and processed for immunofluorescence using standard procedures7 (link). The primary antibodies used for IHC were as follows: sheep anti-αMSH antibody (1:40,000, Millipore, cat #AB5087), rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat #H003-53), rabbit anti-LC3B (1:500, Cell Signaling, cat #2775), rabbit anti-p62 (1:1,000, Abcam, cat #ab64134), rabbit anti-GFP (1:1,000, Invitrogen, cat #A-6455), guinea pig anti-insulin (1:500, Abcam, cat #ab7842), and rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems, cat #139103). The primary antibodies were visualized with Alexa Fluor 568 donkey anti-sheep IgG (1:200, Invitrogen, cat #A21099), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A21206), Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen, cat #A21202), Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A10042), or Alexa Fluor 488 goat anti-guinea pig IgG (1:200, Invitrogen, cat #A11073). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat #H3569) to visualize cell nuclei.
Bis benzamide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bis-benzamide is a chemical compound used in laboratory settings. It functions as a fluorescent dye that binds to DNA, allowing for the visualization and detection of DNA in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Sheep anti αmsh antibody, by Merck Group (3 mentions)
Alexa fluor 568 donkey anti sheep igg, by Thermo Fisher Scientific (3 mentions)
Rabbit anti agrp, by Phoenix Pharmaceuticals (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit anti pomc, by Phoenix Pharmaceuticals (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (1 mentions)
Vacht, by Synaptic Systems (1 mentions)
Alexa fluor 488 goat anti guinea pig igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p62, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
A11011, by Thermo Fisher Scientific (1 mentions)
A11073, by Thermo Fisher Scientific (1 mentions)
A10042, by Thermo Fisher Scientific (1 mentions)
H 029 30, by Phoenix Pharmaceuticals (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab152, by Merck Group (1 mentions)
Sheep anti α msh, by Merck Group (1 mentions)
11 protocols using bis benzamide
1
Immunofluorescence Analysis of Mouse Brain
2
Immunofluorescence Staining of Mouse Brain and Pancreas
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Thirteen- to 14-week-old mice were perfused transcardially with 4% paraformaldehyde. Brains were then frozen, sectioned at 30 μm thickness, and processed for immunofluorescence using standard procedures (24 (link)). The primary antibodies used for IHC were as follows: rabbit anti-POMC (1:20,000, Phoenix Pharmaceuticals catalog H-029-30) and rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals catalog H-003-53). The primary antibodies were visualized with goat anti-rabbit Alexa Fluor 488 (1:200, Thermo Fisher Scientific catalog A11008) or anti-rabbit IgG Alexa Fluor 568 (1:200, Thermo Fisher Scientific catalog A11011) secondary antibodies.
Pancreatic tissues were dissected from perfused animals, frozen, cut at 20 μm thickness, and processed for immunofluorescence using standard procedures (24 (link)). The primary antibodies used for IHC were as follows: guinea pig anti-insulin (1:500, Genetex catalog GTX39371), rabbit anti-VAChT (1:500, Synaptic Systems catalog 139 103), and rabbit anti-TH (1:500, MilliporeSigma catalog Ab152). The primary antibodies were visualized with donkey anti–guinea pig Alexa Fluor 488 (1:200, Thermo Fisher Scientific catalog A11073) or donkey anti-rabbit Alexa Fluor 568 (1:200, Thermo Fisher Scientific catalog A10042) secondary antibodies.
All sections were counterstained with bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei.
Pancreatic tissues were dissected from perfused animals, frozen, cut at 20 μm thickness, and processed for immunofluorescence using standard procedures (24 (link)). The primary antibodies used for IHC were as follows: guinea pig anti-insulin (1:500, Genetex catalog GTX39371), rabbit anti-VAChT (1:500, Synaptic Systems catalog 139 103), and rabbit anti-TH (1:500, MilliporeSigma catalog Ab152). The primary antibodies were visualized with donkey anti–guinea pig Alexa Fluor 488 (1:200, Thermo Fisher Scientific catalog A11073) or donkey anti-rabbit Alexa Fluor 568 (1:200, Thermo Fisher Scientific catalog A10042) secondary antibodies.
All sections were counterstained with bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei.
3
Immunofluorescence Staining of Hypothalamic Neuropeptides
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Sections were processed for immunofluorescence using standard procedures (Bouret et al., 2004b (link); Steculorum et al., 2015 (link); Croizier et al., 2016 (link)). The primary antibodies used for IHC were as follows: sheep anti-α-MSH (1:40,000, Millipore; Steculorum et al., 2015 (link)), sheep anti-NPY (1:3,000, Chemicon; Lee et al., 2013 (link)), rabbit anti-β-endorphin (1:5,000, Millipore; Grayson et al., 2010 (link)), rabbit anti-POMC (1:2,000, Phoenix Pharmaceuticals; MacKay et al., 2017 (link)). The primary antibodies were visualized with Alexa Fluor 568 goat anti-rabbit IgGs or Alexa Fluor 568 donkey anti-sheep IgGs (1:200, Invitrogen). Sections were counterstained using bisbenzamide (1:10,000, Invitrogen), to visualize cell nuclei, and coverslipped with buffered glycerol (pH 8.5).
4
In Situ Hybridization for Npy and Pomc
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Sense and antisense digoxigenin-labeled riboprobes were generated from plasmids containing PCR fragments of Npy and Pomc. Sections were post-fixed in 4% paraformaldehyde and incubated with Proteinase K (Promega). They were incubated in triethanolamine (TEA) and then in TEA containing glacial acid acetic. Sections were pre-hybridized in hybridization buffer and then hybridized with denatured probes (300 ng for Npy and 400 ng for Pomc) overnight at 62 C. After washes in stringency solutions, sections were blocked in TNB solution (Roche) and incubated in a horseradish peroxidase-conjugated sheep anti-DIG antibody (1:400, Roche Applied Sciences; Padilla et al., 2010 (link)). DIG was visualized using a TSA PLUS Biotin Kit (Perkin Elmer). Sections were first incubated in the Biotin Amplification Reagent (1:50), and then in cyanin 2-conjugated streptavidin (1:200, Jackson Immunoresearch). Sections were counterstained using bisbenzamide (1:10,000, Invitrogen) to visualize cell nuclei.
5
Immunofluorescence Analysis of Neuronal Markers
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Anesthetized male mice were perfused transcardially with 4% paraformaldehyde at 2–3 months of age. The brains were then frozen and sectioned at 30-μm-thick and processed for immunofluorescence using standard procedures. The primary antibodies used for immunohistochemistry (IHC) were as follows: rabbit anti-GFP (1:10,000, Invitrogen), rabbit anti-cFos (1:2,000, Oncogene), rabbit anti-p62/SQSTM1 (1:1,000, Abcam), and rabbit anti-ubiquitin (1,000, Dako). The primary antibodies were visualized with Alexa Fluor 488 or Alexa Fluor 568 goat anti-rabbit IgGs (1:200, Invitrogen) and Alexa Fluor 568 donkey anti-sheep IgGs (1:200, Invitrogen). Sections were counterstained using bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei and coverslipped with Fuoromount-G (SouthernBiotech).
6
Leptin-Induced pSTAT3 Activation Assay
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Leptin (3 mg/kg; Peprotech, cat# 450–31) was injected intraperitoneally in P14 pups. Animals were perfused 45 minutes later with a solution of 2% paraformaldehyde. Frozen coronal sections were cut at 30 μm thickness and pretreated for 20 min with 0.5% NaOH and 0.5%H2O2 in KPBS, followed by immersion in 0.3% glycine for 10 min. Sections were then placed in 0.03% SDS for 10 minutes and placed in 4% normal serum + 0.4% Triton X-100 + 1% BSA (fraction V) for 20 minutes before incubation for 48 hours with a rabbit anti-pSTAT3 antibody (1:1,000, Cell Signaling, cat# 9131). The primary antibody was localized with Alexa Fluor 568 Goat anti-Rabbit IgGs (Invitrogen; 1:200, cat# A11036). Sections were counterstained using bis-benzamide (Invitrogen; 1:10,000, cat# H3569) to visualize cell nuclei and cover slipped with buffered glycerol (pH 8.5).
7
Quantifying αMSH-Immunoreactive Fibers in Mouse PVH
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15-17-weeks old male mice (n = 4/group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-um thick, and processed for immunofluorescence using standard procedures (Bouret et al., 2004 (link)). Briefly, sections were incubated with a sheep anti-αMSH antibody (1:40,000, Millipore). The primary antibody was visualized with Alexa Fluor 488 donkey anti-sheep IgG (1:200, Millipore). Sections were counterstained using bis-benzamide (1:10,000, Invitrogen) to visualize cell nuclei. Two sections through various anatomical compartments of the PVH (PVHmpd and PVHpml, neuroendocrine; and postPVH, autonomic) were acquired using a Zeiss LSM 710 confocal system equipped with a 20X objective. For the quantitative analysis of fibers density, each image plane was binarized to isolate labeled fibers from the background and to compensate for differences in fluorescence intensity. The integrated intensity, which reflects the total number of pixels in the binarized image, was then calculated for each image. This procedure was conducted for each image stack.
8
Immunofluorescence Analysis of POMC, αMSH, and AgRP
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Ten- to twelve-week-old mice were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-μm thickness, and processed for immunofluorescence using standard procedures [8 (link),37 ]. The primary antibodies used for IHC were as follows: rabbit anti-POMC (1:20,000, Phoenix Pharmaceuticals, cat# H029-30), sheep anti-αMSH antibody (1:40,000, Millipore, cat# AB5087), and rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat# H003-53). The primary antibodies were visualized with Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Millipore, cat# A21206), Alexa Fluor 568 donkey anti-sheep IgG (1:200, Millipore, cat# A21099), or Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Millipore, cat# A10042). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat# H3569) to visualize cell nuclei.
9
Lipid Accumulation in Hypothalamic Cells
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mHypoE-N43/5 cells were treated with vehicle or a combination of palmitic (250 μM; Sigma, cat# P9767), lauric (1mM; Sigma, cat# W261416), and myristic acids (200 μM; Sigma, cat# M3128) for 24 hours and 2 μM of Bodipy 493/503 (4,4-difluro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene; Invitrogen, cat# D3922) and Alexa Fluor 568 Phalloidin (0.1 μM; Invitrogen, cat# A12380) were added to culture media for 15 minutes at room temperature. mHypoE-N43/5 were then fixed in a solution of 4% paraformaldehyde for 5 minutes and washed with KPBS. Slides were counterstained using bis-benzamide (Invitrogen; 1:10,000, cat# H3569) to visualize cell nuclei.
10
Analysis of Liver Fibrosis and Cell Transplantation
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Liver tissues were removed, post-fixed in 4% PFA overnight, and then embedded in paraffin. For determination of liver fibrosis, 5-μm-thick paraffin sections were deparaffinized and stained with Picro-Sirius red. The ratio of the Sirius red-positive area to the total area was examined using the ImageJ 1.35s software (National Institutes of Health, Bethesda, MD).
For detection of transplanted cells and HGF expression, deparaffinized liver sections were boiled in citrate buffer (pH 6.0) for 10 min. After blocking in 5% normal serum, the sections were incubated with anti-human HGF antibody (R&D Systems, Minneapolis, MN, USA) and human mitochondria-specific antibody (hMt; Chemicon, Temecula, CA, USA) at 4 °C overnight. Sections were then incubated with AlexaFluor 488- or 594-conjugated anti-IgG secondary antibodies (Life Technologies, Grand Island, NY, USA) and counterstained with bis-benzamide (Molecular Probes, Eugene, OR, USA) to visualize nuclei. To detect Ito cells, sections were immunostained with anti-desmin antibody (DAKO, Tokyo, Japan) or anti-α-smooth muscle actin antibody (α-SMA; DAKO) and visualized using an ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Immunostained images were acquired using a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany).
For detection of transplanted cells and HGF expression, deparaffinized liver sections were boiled in citrate buffer (pH 6.0) for 10 min. After blocking in 5% normal serum, the sections were incubated with anti-human HGF antibody (R&D Systems, Minneapolis, MN, USA) and human mitochondria-specific antibody (hMt; Chemicon, Temecula, CA, USA) at 4 °C overnight. Sections were then incubated with AlexaFluor 488- or 594-conjugated anti-IgG secondary antibodies (Life Technologies, Grand Island, NY, USA) and counterstained with bis-benzamide (Molecular Probes, Eugene, OR, USA) to visualize nuclei. To detect Ito cells, sections were immunostained with anti-desmin antibody (DAKO, Tokyo, Japan) or anti-α-smooth muscle actin antibody (α-SMA; DAKO) and visualized using an ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Immunostained images were acquired using a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany).
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