After a minimum of 6 weeks of recovery, the animals were euthanized following AVMA guidelines. The animals were perfused transcardially with heparinized saline followed by a solution of 4% or paraformaldehyde in 0.1M phosphate buffer. For each case, the brain was removed and cryoprotected through an ascending series of glycerol solutions. The cryoprotected tissue was then frozen in isopentane and serially sectioned (at 40 µm) using a sledge microtome. Series with every tenth section (400 µm apart) of free-floating sections were processed for either histochemistry using the ABC Elite Kit (Vector laboratories PK-6100) or via immuno-visualization of ChAT, AChE, NeuN, and CFP or mCherry reporters. Primary Antibodies: anti-CFP rabbit polyclonal (Abcam ab290), goat polyclonal anti-ChAT (Millipore AB144P), rabbit polyclonal anti-mCherry (Abcam ab167453), goat anti-mouse Alexa 647 (Invitrogen A21235), mouse anti-AChE (Ivitrogen MA3-042). Secondary Antibodies: biotinylated anti-rabbit (Vector laboratories BA1000), goat anti-mouse Alexa 647 (Invitrogen A21235), goat anti-rabbit Alexa 555 (Invitrogen A21428), goat anti-chicken Alexa 488 (Invitrogen A11039), and goat anti-rabbit Alexa 488 (Invitrogen A11008).
Goat anti mouse alexa 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse Alexa 647 is a secondary antibody conjugated with the Alexa Fluor 647 fluorescent dye. It is designed to bind to mouse primary antibodies, allowing for the detection and visualization of target proteins or molecules in various lab applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (10 mentions)
Goat anti rabbitalexa647, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Goat anti chicken alexa 488, by Thermo Fisher Scientific (3 mentions)
Fluoromount g mounting medium, by Electron Microscopy Sciences (3 mentions)
Dapi no 18860, by Serva Electrophoresis (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
A 21244, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse alexa 546, by Thermo Fisher Scientific (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Goat anti mouse alexa 568, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa 555, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa568, by Thermo Fisher Scientific (2 mentions)
Goat anti ratalexa647, by Thermo Fisher Scientific (2 mentions)
V9131, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ab17056, by Abcam (2 mentions)
34 protocols using goat anti mouse alexa 647
1
Transcardial Perfusion and Cryosectioning
2
Quantitative Dysferlin Analysis in Muscle Tissue and Cells
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Transversal muscle tissue cryosections of 6 μm were fixed for 5 min in acetone at −20°C and blocked with 1% BSA in PBS for 45 min at room temperature. Sections were incubated overnight at 4°C with the primary ROMEO anti-Dysferlin rabbit monoclonal antibody (Abcam, Cambridge, UK, 1:100). Sections were incubated with the secondary goat anti-rabbit Cy3-conjugated antibody (Dianova, Hamburg, Germany, 1:500) for 45 min at room temperature. Nuclei were stained with Hoechst. The sections were mounted with AquaMount (Polysciences, Hirschberg, Germany). Cells were seeded on glass-bottom plates (ibidi, Martinsried, Germany) and fixed with 3.7% formaldehyde for 10 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. Cells were blocked with 1% BSA in PBS for 1 hr at room temperature and incubated overnight at 4°C with two primary antibodies: mouse monoclonal antibody anti-Desmin (Dako, Jena, Germany; 1:100) and rabbit monoclonal antibody ROMEO anti-Dysferlin (Abcam, 1:150). Cells were incubated for 1 hr at room temperature with the following secondary antibodies (both diluted to 1:500): polyclonal Alexa 568 donkey anti-rabbit (Life Technologies) and polyclonal Alexa 647 goat anti-mouse (Life Technologies). Nuclei were stained with Hoechst.
3
Immunohistochemistry for Cytoskeletal Markers
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Immunohistochemistry was performed according to Sive et al. (2000) using primary antibodies against monoclonal α-tubulin (1:1000, Sigma-Aldrich T9026), monoclonal gamma-tubulin (1:1000, Sigma-Aldrich T6557), polyclonal Serotonin (5HT) (1:500, Merck Millipore AB938), Alexa Fluor 488 phalloidin (1:1000, Life Technologies A12379). Alexa 647 goat anti-rabbit (1:250, Life technologies A21244) and Alexa 647 goat anti-mouse (1:500, Life Technologies A21235) were used as secondary antibodies. Samples were imaged using Leica TCS SPE confocal microscope. Images were analyzed using LAS AF Lite, ImageJ (NIH) and Photoshop (Adobe).
4
Quantifying ALCAM and Proliferation in Tissue Microarray
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Immunofluorescence (IF) was performed on the tissue microarray described above. Sections (5μm) were deparaffinized and rehydrated. Antigen retrieval was performed by pressure cooker in citrate buffer (pH 6.0) and sections blocked in 20% Aquablock (East Coast Biologics) plus 0.05% Tween-20. IF was performed with primary antibodies mouse anti-ALCAM (MOG/07; 1:100; NovocastraTM, Leica Biosystems), rabbit anti-Ki67 (Clone SP6; 1:500; Thermo Scientific), and Hoechst 33342 as well as, secondary antibodies Alexa-546 goat anti-rabbit and Alexa-647 goat anti-mouse (1:500; LifeTechnologies). Collagen was stained with Alexa 488-conjugated CNA35 (gift from Erin Rericha, Vanderbilt) [46 (link), 47 (link)]. IF slides were mounted in ProLong Gold Antifade reagent (Invitrogen). Fluorescence intensity and thresholded area were quantified in the epithelium in each TMA core with an Image J-based batch macro. Collagen staining was used to distinguish between the epithelial, stromal and muscular compartments. Hoechst was used to define the nuclear compartment while Ki67 marked proliferating cells. Percent thresholded area of ALCAM was subsequently used for analysis.
5
Immunofluorescence Analysis of Mouse Testis
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Immunofluorescence labeling of frozen sections of mouse testis was performed using rabbit polyclonal anti UPF1, rabbit polyclonal anti UPF2 [35 (link)], rabbit polyclonal anti MVH (Abcam), mouse monoclonal anti SYCP3 [57 (link)], guinea pig polyclonal anti C-term TDRD6 (this study). Sections were fixed using 4% PFA for 20 minutes, blocked and permeabilized with 2% BSA, 0.1% Triton-X100 in PBS and incubated overnight with primary antibodies. Slides were washed with PBST and probed for 2 h with secondary antibodies Alexa-566-labeled goat anti guinea pig, Alexa-488-labeled goat anti rabbit, or Alexa-647-goat anti mouse (Molecular probes, Invitrogen). For double immunostaining rabbit polyclonal antibodies were labeled using the Zenon Rabbit IgG Labeling Kits (Molecular Probes, Invitrogen). Slides washed again with PBST and nuclei were visualized with DAPI. Images acquired with a Zeiss LSM 510 confocal microscope and quantification of signal intensity was done with ImageJ.
6
Immunofluorescence Staining of Cell Adhesion Proteins
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Cells were grown onto 8 mm diameter coverslips, transfected and fixed in 3.7% formaldehyde solution (47608, Fluka, Loughborough, UK) at room temperature for 15 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 and blocked with 10% horse serum to prevent non-specific staining. The primary antibody was then added to the blocking buffer and the cells incubated overnight at 4 °C. Primary antibodies: Mouse anti-γ-catenin (610254, BD Transduction), mouse anti–p120 (P17920, BD Biosciences), mouse anti-α-tubulin (T9026, Sigma-Aldrich), and E-cadherin (ECCD2, Zymed laboratories). The day after, the cells were washed in PBS with 0.1% Triton X-100 for three times and incubated with Alexa 546 goat anti-mouse, Alexa 647 goat anti-mouse, (Molecular Probes, Eugene, OR, USA) fluorophore conjugated secondary antibodies for signal detection. The actin filaments were stained using Alexa 546-conjugated phalloidin (Molecular Probes) added after the blocking step. After washes in PBS with 0.1% triton, coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold Antifade Reagent (Molecular Probes). The micrographs were obtained at Confocal Spectral Leica TCS SP5 and LAS-AF 1.8.1 Leica software.
7
Elastase Treatment Effects on Disc Elastin
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To visualize the effect of elastase treatment, three pairs of disc were used for immunohistochemistry and included in the PBS or elastase treatment. Porcine arteries were used as positive controls for elastin staining. Following treatment, samples were washed with PBS, fixed overnight in 4% paraformaldehyde, and the central, lateral and medial parts of the disc were cut into two parts, one used for transverse and the other for sagittal sectioning. Then, each part was sectioned at 10 μm.
The sections were incubated with PBS followed by blocking buffer (1% BSA, 20% goat serum, PBS) for 20 min and incubated with a cocktail of primary antibodies containing rabbit polyclonal anti‐collagen I (1:1000, ab34710, Abcam, MA) and mouse polyclonal anti‐elastin (1:100, ab9519, Abcam, MA) overnight at 4°C. Thereafter, sections were washed with PBS, and a cocktail of secondary antibodies containing Alexa 488 goat anti‐rabbit (1:2000; Invitrogen, CA) and Alexa 647 goat anti‐mouse (1:200; Invitrogen, CA) was added for 1 hr. Then, sections were washed with PBS, stained with DAPI for 10 min, washed in PBS and mounted with Vectashield mounting medium (Vector laboratories, CA). Images were obtained with an inverted confocal microscope (Leica SP8, Germany).
The sections were incubated with PBS followed by blocking buffer (1% BSA, 20% goat serum, PBS) for 20 min and incubated with a cocktail of primary antibodies containing rabbit polyclonal anti‐collagen I (1:1000, ab34710, Abcam, MA) and mouse polyclonal anti‐elastin (1:100, ab9519, Abcam, MA) overnight at 4°C. Thereafter, sections were washed with PBS, and a cocktail of secondary antibodies containing Alexa 488 goat anti‐rabbit (1:2000; Invitrogen, CA) and Alexa 647 goat anti‐mouse (1:200; Invitrogen, CA) was added for 1 hr. Then, sections were washed with PBS, stained with DAPI for 10 min, washed in PBS and mounted with Vectashield mounting medium (Vector laboratories, CA). Images were obtained with an inverted confocal microscope (Leica SP8, Germany).
8
Immunofluorescence Labeling of Cells
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After fixation the cells were permeabilized with 0.1% Triton X (American Bioanalytical) for 10 minutes. They were washed 3 times with PBS. The cells were blocked with 3% IgG free BSA (Accurate Chemicals). The primary HA antibody (Covance) was diluted 1:1000 in 3% BSA. Subsequently, the cells were incubated with this solution for 1 h at room temperature. The cells were then washed 3 times with 3% BSA and labeled with the secondary antibody for 30 minutes at room temperature. Depending on the original live cell staining, the secondary antibody was either Alexa 546 goat anti-mouse (Halo/SNAP-tag labeling with SiR) or Alexa 647 goat anti-mouse (Halo/SNAP-tag labeling with TMR) (Invitrogen). The secondary antibody was diluted 1:1000 in 3% BSA. Subsequently the cells were washed 3 times with PBS and once with water. Next, they were mounted onto glass microscopes slides with ProLong® Gold Antifade (Life Technologies). The slides were protected from light overnight at room temperature before imaging.
9
Immunofluorescence Analysis of FAK and Paxillin
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HUVECs (6 × 104 cells) were cultured on 0.2% gelatin-coated coverslips for 48 h. Cells were starved in basal medium containing 0.5% FCS for 16 h before treatment with conditioned medium. Cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 in PBS, and incubated in 3% BSA in PBS for 1 h. pY397FAK, vinculin, or pY118 paxillin antibodies (1:100 in blocking solution) were incubated overnight at 4°C. Coverslips were washed in PBS with 0.2% Tween-20, incubated with Alexa-488 goat anti-rabbit or Alexa-647 goat anti-mouse (Invitrogen) for 30 min. After a second washing step, samples were incubated with Alexa Fluor 546-conjugated phalloidin (Invitrogen) for 30 min. Images were acquired using a Zeiss LSM700 confocal microscope.
10
Quantifying Erythrocyte Protein Expression
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40μl of washed human and rat RBCs (5x108 cells) were fixed overnight in 1mL of paraformaldehyde 4% at 4°C. For permeabilization, after washing with staining buffer (PBS with 1% FBS, 0.09% NaN3), the cell pellet was suspended in 0.5mL of 0.1% Triton for 10 minutes. After one wash to remove Triton X-100, cells were incubated in FBS 10% for 15 minutes at 4°C. Erythrocytes were incubated with 2.5μg of mouse anti-eNOS (610297, BD Biosciences) (1 hour) and 1μg of polyclonal rabbit anti-Aquaporin-1 (AB2219, Millipore) (20 minutes) antibodies at room temperature (RT). The secondary antibodies Alexa-647 goat anti-mouse and Alexa-488 goat anti-rabbit (Invitrogen) were incubated for 20 minutes at RT. Negative controls were obtained by incubating only with secondary antibodies and with mouse non-specific IgG1,k Alexa-647-conjugated (Invitrogen). Finally, RBCs were suspended in staining buffer. Data acquisition was performed with a FACSCantoII™ flow cytometer (BD Biosciences) and data analysis with FlowJo software (BD Biosciences).
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