Non-neurological control and C9orf72 patient postmortem paraffin embedded motor cortex sections were obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 5 : Supplemental Table 2 for demographic information). Antigen retrieval and immunofluorescent staining was conducted as previously described [3 (link)]. Antibodies for immunostaining are as follows: 1:500 Rabbit Anti-Lamin B1 (Abcam ab16048), 1:1000 Guinea Pig Anti-Map2 (Synaptic Systems 188,004), 1:1000 Goat Anti-Guinea Pig Alexa 488 (Invitrogen A11073), 1:1000 Goat Anti-Rabbit Alexa 647 (Invitrogen A21245). Nuclei from Map2 positive Layer V neurons were imaged with a 63X objective and a Zeiss Axioimager Z2 fluorescent microscope equipped with an apotome2 module. All images were acquired using identical exposure times. Images are presented as default apotome processed images generated in Zeiss Zen Blue 2.3.
Goat anti rabbitalexa647
Manufactured by Thermo Fisher Scientific
Goat anti-Rabbit Alexa647 is a secondary antibody conjugated with Alexa Fluor 647 dye, which is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging techniques. This product provides a reliable and sensitive tool for researchers to identify and localize target proteins or cellular structures labeled with rabbit primary antibodies.
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Lab products found in correlation
Goat anti mouse alexa 647, by Thermo Fisher Scientific (4 mentions)
Fluoromount g mounting medium, by Electron Microscopy Sciences (3 mentions)
Dapi no 18860, by Serva Electrophoresis (3 mentions)
Goat anti mouse alexa 555, by Thermo Fisher Scientific (2 mentions)
Goat anti ratalexa647, by Thermo Fisher Scientific (2 mentions)
Goat anti chicken alexa 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Goat anti guinea pig alexa 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti lamin b1, by Abcam (1 mentions)
Guinea pig anti map2, by Synaptic Systems (1 mentions)
Zen blue 2, by Zeiss (1 mentions)
Axio imager z2 fluorescent microscope, by Zeiss (1 mentions)
Goat anti mouse alexa 568, by Thermo Fisher Scientific (1 mentions)
Fm 1 43fx, by Thermo Fisher Scientific (1 mentions)
Anti brdu antibody, by BD (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Dulbecco s pbs dpbs, by Thermo Fisher Scientific (1 mentions)
Aiib2, by Merck Group (1 mentions)
12 protocols using goat anti rabbitalexa647
1
Imaging Motor Cortex Neurons in C9orf72 ALS
2
Whole mount immunolabeling of inner ear tissues
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Whole mount immunolabeling was performed using established protocols (Ungos et al., 2003).54 (link) The primary antibodies used were: anti-Otoferlin antibody (mouse monoclonal, 1:200, DSHB, University of Iowa), anti-Sox 2 antibody (rabbit polyclonal, 1:100, Invitrogen) and anti-BrdU antibody (mouse monoclonal, 1:100, BD Biosciences). Secondary antibodies used were: goat anti-rabbit Alexa 647 (1:1000, Invitrogen), goat anti-mouse Alexa 568 (1:1000, Invitrogen), and goat anti-mouse Alexa 647 (1:1000, Invitrogen). Mature hair cells were labeled by a 1-min incubation in 3μM FM1-43FX (Invitrogen).55 (link) Nuclei were labeled with 30μM DAPI (Thermo Fisher).
3
Immunostaining of H. pylori-infected cells
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One day prior to experiments cells were seeded at 5 x 104 cells in a 24-well plate equipped with uncoated cover slides and grown overnight at 37°C and 5% CO2. Cells were infected with Hp wild type or isogenic mutant strains with an MOI of 10 for 3h at 37°C and 5% CO2. For immunostaining cells were fixed with 4% PFA for 10 min at room temperature. Cells were washed twice with Dulbecco´s PBS (DPBS, Life Technologies) and blocked overnight with 2% FCS in PBS at 4°C. Fixed cells were incubated with mouse anti-CEACAM5 (26/3/13, Genovac, 1:300), rabbit anti-Hp (AK175, 1:400) and rat anti-integrin beta1 (AIIB2, Millipore, 1:200) for 1h at room temperature. After washing secondary antibodies were applied (goat anti-rat Alexa488, goat anti-mouse Alexa555 and goat anti-rabbit Alexa647 all from Invitrogen, 1:1000) and incubated for 1h at room temperature in the dark. Cell nuclei were stained with DAPI (5μg/ml) for 10 min. Samples were mounted on the cover slip with Fluorescent Mounting Medium (DAKO). A cytospin3 (Shandon) was used to centrifuge suspension cells onto glass slides. Micrographs were taken with a confocal laser scanning microscope (LSM880, Zeiss) with Airyscan Module using a 63x oil immersion objective.
4
Immunofluorescent Staining of BMDMs
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Day 3 BMDMs were seeded onto RetroNectin-coated chamber slides (Takara Bio Inc.) and stimulated as described in figure legends. Cells were then fixed with 4% paraformaldehyde in PBS for 30 min and washed twice in PBS before blocking and permeabilization in blocking buffer (PBS, 10% FCS, and 0.5% Triton X-100) for 60 min. Staining was then performed with primary antibodies ASC (1:500 N-15; Santa Cruz Biotechnology, Inc.) or phalloidin-FITC (1:400; Sigma-Aldrich) in blocking buffer overnight at 4°C. Cells were washed three times with blocking buffer, and secondary antibody staining for ASC was performed with goat anti–rabbit Alexa 647 (1:1,000; Invitrogen) in blocking buffer for 60 min at room temperature (in the dark). Cells were washed twice with PBS before imaging. Images were acquired with a confocal microscope (LSM 780; Carl Zeiss) at room temperature using a 40× oil objective with Immersol 518 F (refractive index n = 1.518; Carl Zeiss) and a 1.4 numerical aperture and were acquired with ZEN 2012 v8.1 software (Carl Zeiss). Image channels were merged and converted to TIFF using FIJI software.
5
Immunocytochemical Analysis of PDE4D Localization
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To investigate PDE4D protein localization, HT22 cells were seeded on 12 mm glass coverslips (VWR, 631–1577) coated with 100 μg/mL Poly-l -Ornithine (Sigma, P4957) and 1 μg/mL laminin (Sigma, L2020) and grown for 24 h. After fixation in 4% paraformaldehyde, cells were permeabilized using 0.1% Triton X-100. After blocking with 10% BSA for 1 h, cells were incubated with rabbit anti-PDE4D (1:250; ab14613, Abcam) overnight at 4 °C. Then, cells were incubated with goat anti-rabbit Alexa647 (1:250; Invitrogen) for 1 h at room temperature. Finally, nuclei were counterstained with Hoechst (1:500; Sigma).
For immunocytochemistry following transfection experiments in HT22, the same protocol was used except for the antibodies used. Mouse anti-FLAG primary antibodies (1:1000; M2 clone, Sigma-Aldrich) and donkey anti-mouse Alexa488-conjugated secondary antibody (1:250; Invitrogen) were used to determine which cells were successfully transfected and expressed the FLAG-encoding PX458 plasmid. PDE4D localization was imaged after mounting the coverslips on microscope glasses using a disk spinning unit (DSU) microscope (Olympus). Morphology assessment of transfected, FLAG-positive HT22 cells was performed as described above.
For immunocytochemistry following transfection experiments in HT22, the same protocol was used except for the antibodies used. Mouse anti-FLAG primary antibodies (1:1000; M2 clone, Sigma-Aldrich) and donkey anti-mouse Alexa488-conjugated secondary antibody (1:250; Invitrogen) were used to determine which cells were successfully transfected and expressed the FLAG-encoding PX458 plasmid. PDE4D localization was imaged after mounting the coverslips on microscope glasses using a disk spinning unit (DSU) microscope (Olympus). Morphology assessment of transfected, FLAG-positive HT22 cells was performed as described above.
6
Multiparameter Flow Cytometry Analysis
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Cells were harvested, stained, acquired on a FACSCantoII (Becton Dickinson) and analyzed with FlowJo (Treestar). Dead cells were excluded using the Fixable Viability Dye eFluor506 (eBioscience). Antibodies conjugated to FITC, PE, PerCP Cy5.5, PE Cy7, APC, APC-Cy7 or Pacific Blue were purchased from BD Biosciences, eBioscience, BioLengend and R&D Systems. PE- or APC- conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) were obtained from the tetramer facility of the US National Institutes of Health. In brief, cells were incubated with FC block and stained with antibodies, and then fixed with 2% paraformaldehyde. For PLZF, T-bet, and TCF-1 intracellular staining, cells were permeabilized and stained with anti-PLZF (D-9) (Santa Cruz Biotechnology, Inc.) plus FITC anti-mouse (BD Biosciences), anti-T-bet PerCP-Cy5.5 (eBio4B10), both purchased from eBioscience, and with anti-TCF-1 (C63D9) (Cell Signaling) followed by goat anti-rabbit-Alexa647 (Invitrogen), using the Foxp3 Staining Buffer kit (eBioscience).
7
Immunostaining of Drosophila Gut
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Guts were dissected in PBS and transferred into 4% PFA immediately after dissection and staining was performed on an orbital shaker. After 45 min of fixation guts were washed once with PBS for 10 min. Antibodies were diluted in 0.5% PBT + 5% normal goat serum. The incubation with primary antibodies (1:250 anti-Dlg-1 [mouse; Developmental studies Hybridoma Bank (DSHB)]; 1:50 anti-Pros [mouse; DSHB]; 1:2000 anti-pH3 [rabbit; Merck Millipore, 06–570]; 1:50 anti-EcR common Ag10.2 [mouse; DSHB]; 1:500 anti-HA High Affinity 3F10 [rat; Merck, Sigma-Aldrich]; 1:1500 anti-ß-Galactosidase preabsorbed [rabbit; Cappel Research Products]) was performed at 4°C over night. After washing with PBS guts were incubated with secondary antibodies (1:500 Goat anti-MouseAlexa647 [Invitrogen]; 1:500 Goat anti-RatAlexa647 [Invitrogen]; 1:500 Goat anti-RabbitAlexa647 [Invitrogen]) and DAPI (1:1000; 100 µg/ml stock solution in 0.18 M Tris pH 7.4; DAPI No. 18860, Serva, Heidelberg) for at least 3 hr at RT. Guts were washed a last time with PBS prior to mounting in Fluoromount-G Mounting Medium (Electron Microscopy Sciences).
8
Multiparametric Analysis of Cell Surface Markers
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Cells were analyzed with a 4-laser Attune NxT Acoustic Cytometer (Thermo Fisher Scientific). Samples were resuspended in FLOW buffer (1x PBS; 3 mM EDTA (v/v); 3% FBS (v/v)), and the following fluorescently tagged antibodies were used to label cells for 30 minutes at 4°C: mouse monoclonal anti-human CD133-PE (1 : 20, cat no. 130-110-962, Miltenyi), mouse monoclonal anti-human CD44-PE (1 : 20, cat no. 130-095-180, Miltenyi), and mouse monoclonal anti-human EpCAM-APC (1 : 20, cat no. 130-113-260, Miltenyi). For ANTXR1 detection, cells were first labeled with rabbit anti-human TEM8 (1 : 50, cat no. ab21270, Abcam), washed three times with 1x PBS and subsequently incubated with goat-anti-rabbit (Alexa 647 1 : 500, cat no. A27040, Invitrogen). DAPI was used to mark and exclude dead cells, and data were analyzed using the software FlowJo v9.3 (Tree Star Inc., Ashland, OR). Autofluorescent cells were excited with blue laser 488 nm and selected as the intersection with the filters 530/40 and 580/30 as previously described [14 (link)].
9
Dissected Fly Gut Immunohistochemistry
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Dissected guts from mated female flies were fixed in 4% PFA in 1XPBS for 45 min. After fixation the guts were washed with 1XPBS for 10 min and stained with primary antibodies 1:500 anti-Ssk (rabbit; [84 (link)]); 1:250 anti-Dlg-1 [mouse; Developmental studies Hybridoma Bank (DSHB)]; 1:250 anti-Pros [mouse; Developmental studies Hybridoma Bank (DSHB)], 1:200 anti-Casp3 [rabbit, Cell signalling technology cleaved caspase-3 (Asp175)], 1:200 anti-GFP [chicken, Abcam (ab13970)], 1:200 anti-Dl [mouse, Developmental studies Hybridoma Bank (DSHB, C594.9B)]) diluted in 0.5% PBT (0.5% Triton (Sigma-Aldrich) in 1XPBS) + 5% normal goat serum (Thermo Fisher Scientific, Berman, Germany). Primary antibody staining was performed at 4 °C over night on an orbital shaker. Next, guts were washed with 1XPBS for 10 min and incubated with secondary antibodies (1:500 Goat anti-RabbitAlexa568 [Invitrogen], 1:500 Goat anti-MouseAlexa647 [Invitrogen], 1:500 Goat anti-RabbitAlexa647 [Invitrogen], 1:500 Goat anti-chickenAlexa488 [Invitrogen]) and DAPI (1:1000; 100 µg/mL stock solution in 0.18 M Tris pH 7.4; DAPI No. 18860, Serva, Heidelberg) for at least 1.5 h at RT. After washing with 1XPBS for 10 min the stained guts were mounted in Fluoromount-G Mounting Medium (Electron Microscopy Sciences).
10
Immunohistochemistry for GSK3β and β-catenin
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Animals were killed by transcardiac perfusion with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (w/v) in deep ketamine/xylazine anesthesia. The brains were removed and post-fixed in PBS containing 4% paraformaldehyde over night before cutting 60-μm-thick coronal sections on a vibratome (VT 1000 S from Leica, Wetzlar, Germany). Floating sections were permeabilized with 2% Triton X-100 in PBS over night at room temperature. For anti-GSK3β and β-catenin staining, sections were blocked with I-Block reagent (2 mg ml−1; Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany). Primary antibodies (GSK3β, H-76, rabbit polyclonal, Santa Cruz Biotechnology, Heidelberg, Germany; β-catenin ab6302, rabbit polyclonal, Abcam, Cambridge, UK) were incubated overnight at room temperature in a 1:100 dilution, according to the manufacturer's recommendations. Slices were washed 3 × 10 min with PBS and then incubated with the secondary antibodies (1:200; goat anti-rabbit Alexa 647, Invitrogen, Life Technologies GmbH) for 5 h. After 3 × 10-min washing in PBS, sections were incubated for 30 mins with anti-YFP Alexa 488 antibodies (Invitrogen, Life Technologies GmbH). Sections were finally washed for 5 × 10 min with PBS before mounting them on glass coverslips with Dako fluorescence conserving media (Dako, Hamburg, Germany).
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