The urethra was harvested and fixed in 10% formalin, embedded in paraffin, and sectioned (5 µm) at the level of the mid-urethra. The sections were then deparaffinized and incubated with mouse monoclonal anti-α smooth muscle actin (ASMA) antibody (1:500 dilution, DakoCytomation, Glostrup, Denmark) or mouse monoclonal anti-sarcomeric actin antibody (1:500 dilution, DakoCytomation) overnight. After washing three times with PBS, the samples were incubated with Alexa 594-labeled goat anti-mouse IgG antibody (1:500 dilution, Invitrogen, Carlsbad, CA, USA), or Alexa 488-labeled goat anti-mouse IgG antibody (1:500 dilution, Invitrogen) for 1 h at room temperature. After washing with PBS, nuclei were stained with 5 mg/mL Hoechst 33342 for 30 min at room temperature. Staining was visualized and photographed with a fluorescence microscope (Nikon Eclipse TE 2000-U; Nikon, Tokyo, Japan). For the quantitative analysis, sections of the mid-urethra at the level of the external striated muscle layer with maximum transverse diameter were selected in each sample. Photomicrographs of 5 fields per section chosen at random were taken at 200 × magnification. The mean ASMA-positive area (× 10–4 µm2) and the mean striated muscle diameter (µm) were measured using a computerized digital morphometric analysis system (Adobe Photoshop CS3 extended, Adobe, San Jose, CA, USA).
Alexa 488 labeled goat anti mouse igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa 488-labeled goat anti-mouse IgG antibody is a secondary antibody used in immunological assays. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies, and the Alexa 488 fluorescent label enables visualization and detection of the bound antibodies.
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Lab products found in correlation
Eclipse te2000 u, by Nikon (1 mentions)
Lsm700 2ch urgb, by Zeiss (1 mentions)
Axio observer z1 bio, by Zeiss (1 mentions)
Anti p53 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti inos, by Merck Group (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Tris base, by Merck Group (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti c parp, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Gadph, by Santa Cruz Biotechnology (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using alexa 488 labeled goat anti mouse igg antibody
1
Urethral Smooth Muscle Morphometry
2
Immunofluorescence Analysis of p53 in Liver Cancer Cells
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After 20 μM GGA or ethanol treatment, HuH-7 and PLC/PRF/5 cells on a glass insert in a 24-well plate were rinsed with PBS(−) and fixed for 40 min with 4% paraformaldehyde containing 2% sucrose in PBS(−) and then rinsed with PBS(−). The cells were permeated with 0.5% TritonX-100 and blocked with 10% FBS. The cells were then incubated at 4°C overnight with a monoclonal anti-p53 antibody (Cell Signaling Technology), followed by a 2.5-h incubation with an Alexa-488-labeled goat anti-mouse IgG antibody (Invitrogen, Molecular Probes, Tokyo, Japan). After rinsing with PBS(−), the cells were mounted in ParmaFluor (Beckman Coulter, Brea, CA, USA), covered on a slide glass, and observed under a confocal laser-scanning fluorescence microscope, an LSM700 2Ch URGB equipped with Axio Observer Z1 Bio (Carl Zeiss, Göttingen, Germany).
3
Immunofluorescence Analysis of IRF5 Translocation
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Cell culture plates and slides were coated with the cardiac extracellular matrices (20 μg/ml, final concentration) described above. Myocyte cultures were treated with or without IRF5D (50 μg/mL, 24 h). IRF5 translocation from the cytosol to the nucleus was determined on cultured myocytes using immunofluorescence confocal microscopy. Anti-IRF5 antibody (Santa Cruz Biotechnologies) was used as the primary antibody and Alexa 488-labeled goat anti-mouse IgG antibody (Molecular Probes) was used as the secondary antibody and DAPI as a nuclear stain. Fluorescent images were captured with a fluorescent Nikon Eclipse microscope and fluorescence intensity in the cytosol and nuclei of the cells in the captured images was measured using NIKON Element imaging software as previously described [12 (link)].
4
Immunofluorescence Analysis of Liver Oxidative Damage
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The liver tissues were fixed in buffered zinc formalin, embedded in paraffin, and sectioned. The localization of 8-nitroguanine (8-NG) and inducible nitric oxide synthase (iNOS) was assessed by double immunofluorescence labeling.[17 (link)] The paraffin sections were incubated with the anti-8-nitroguanine antibody (1 μg/mL) and anti-iNOS (1:300; Sigma, St Louis, MO, USA) overnight at room temperature. To confirm 8-nitroguanine formation in genomic DNA, we pretreated the indicated tissue sections with 10 μg/mL RNase at 37°C for 1 h as previously described.[18 (link)] Sections were then incubated for 3 h with an Alexa 594-labeled goat anti-rabbit IgG antibody and an Alexa 488-labeled goat anti-mouse IgG antibody (both 1:400; Molecular Probes, Eugene, OR, USA). Stained sections were examined using an inverted Laser Scan Microscope (LSM 410; Zeiss, Gottingen, Germany). For the expression of proliferating cell nuclear antigen (PCNA) and signal transducer and activator of transcription 3 (STAT3), immunohistochemistry was performed using an anti-PCNA monoclonal primary antibody (PC10; DAKO, Carpinteria, CA) and an anti-STAT3 monoclonal primary antibody (79D7; Cell Signaling, Danvers, MA), respectively, and Envision+ kits according to the manufacturer’s instructions (DAKO).
5
Molecular Mechanisms of ATD Treatment
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Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin–streptomycin–neomycin (PSN), and trypsin–EDTA were purchased from Gibco Ltd. (Grand Island, NY, USA). Primary antibodies against p53(DO-1), p53(Pab-240), CDK2, Bax, Bcl-2, Bcl-xL pro-caspase 3/8, STAT3, MVK, GADPH, and actin were obtained from Santa Cruz Biotechnology (St. Louis, MO, USA). Anti-cyclin D1, anti-c-PARP, and anti-p-STAT3 (Tyr750) were obtained from Cell Signaling Technology (Beverly, MA, USA). Alexa 488-labeled goat anti-mouse IgG antibody was from Thermo Fisher Scientific, Waltham, MA, USA. ATD was provided by Dr. Lee and synthesized from 5-bromo-2-hydroxy-3-methoxybenzaldehyde, as previously reported [23 (link)]. Tris base and all other materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
6
Immunofluorescence Assay for p53
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After ATD or DMSO treatment, Huh7, PLC/PRF/5, and HepG2 cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. The cells were permeated with 0.1% Triton X-100, then incubated at 4 °C overnight with a monoclonal anti-p53 (Pab-240) antibody, followed by a 1 h incubation with an Alexa 488-labeled goat anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). After washing with PBS containing 0.1% tween 20, the DAPI was added for 10 min. The cells were observed under a fluorescence microscope at 400× magnification.
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