The cells were washed with DPBS and fixed at room temperature for 15 min with 4% paraformaldehyde (Merck Millipore), and washed with DPBS. The fixed cells were blocked for a minimum of 30 min in a blocking solution consisting of KPBS with 0.25% triton-X100 (Fisher Scientific) and 5% donkey serum. The primary antibody (rabbit anti-FOXG1, 1:50 dilution, Abcam, RRID: AB_732415; mouse anti-OCT3/4, 1:500, Santa Cruz Biotechnology, RRID: AB_628051; mouse anti-MAP2, 1:1000, Abcam, RRID: AB_2138153; rabbit anti-PAX6, 1:1000, Biolegend, RRID: AB_2565003; mouse anti-NEUN, 1:1000 dilution, Millipore, RRID: AB_2298772; rabbit anti-TBR1, 1:500, Abcam, RRID: AB_2200219) was added and incubated at room temperature overnight. On the following day, the cells were washed with KPBS and blocked for at least 10 min in donkey serum blocking solution. The secondary antibody (donkey anti-rabbit Cy3, donkey anti-rabbit Cy2, donkey anti-mouse Cy2; 1:200; Jackson Lab) was added with the nuclear stain DAPI (1:1000; Sigma-Aldrich) and incubated at room temperature for approximately 1 h, followed by 2–3 rinses with KPBS. The immunocytochemically labelled cells were then visualized with a Leica microscope (model DMI6000 B), and images were cropped and adjusted in Adobe Photoshop CC 2015.
Donkey anti mouse cy2
Manufactured by Jackson ImmunoResearch
Sourced in China, Cameroon, United States
Donkey anti-mouse Cy2 is a secondary antibody conjugated with the Cy2 fluorescent dye. It is designed to detect and visualize primary antibodies raised in mouse.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit cy3, by Jackson ImmunoResearch (5 mentions)
Mouse anti neun, by Merck Group (3 mentions)
Cy2 donkey anti rabbit, by Jackson ImmunoResearch (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (2 mentions)
Dapi dilactate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti trpv1, by Santa Cruz Biotechnology (2 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Dapi nuclear stain, by Merck Group (1 mentions)
Rabbit anti foxg1, by Abcam (1 mentions)
Mouse anti oct3 4, by Santa Cruz Biotechnology (1 mentions)
Mouse anti map2, by Abcam (1 mentions)
Rabbit anti pax6, by BioLegend (1 mentions)
Rabbit anti tbr1, by Abcam (1 mentions)
Dmi6000 b, by Adobe (1 mentions)
Donkey anti rat cy5, by Jackson ImmunoResearch (1 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Gata4, by Thermo Fisher Scientific (1 mentions)
Dm5500 mz16f, by Leica (1 mentions)
Itga4, by BD (1 mentions)
10 protocols using donkey anti mouse cy2
1
Immunocytochemical Staining Protocol
2
Immunofluorescence Analysis of Mouse Embryos
3
Dual-Label Immunostaining for BrdU and IdU
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Prior to immunostaining, slides were washed twice with ddH2O for 5 min, 1 × 2.5 M HCl, denatured with 2.5 M HCl for 1 h, rinsed twice with PBS and then washed with blocking solution (PBS with 1% BSA and 0.1% Tween-20) twice for 5 min and then for 1 h. For immuno-labelling all antibodies were dissolved in blocking solution. Slides were then incubated with a rat anti-BrdU antibody (BU1/75 [ICR1], 1:500, Abcam cat# 6326; RRID: AB_305426) to detect BrdU for 1 h under humidified conditions, rinsed with PBS (× 3), fixed for 10 min with 1% formaldehyde, rinsed with PBS (× 3), and quenched with glycine. Slides were then rinsed with PBS (× 3) followed by overnight, 4 °C incubation with mouse anti-BrdU (B44, 1:100, BD Biosciences cat#347580; RRID: AB_400326) to detect IdU. Slides were then washed twice with PBS, 3 times for 5 min in blocking solution, followed by incubation in the appropriate fluorescently-conjugated secondary antibodies diluted in blocking solution (1:500; donkey anti-rat Cy3 cat#712–165-153 RRID: AB_2340667; donkey anti-mouse Cy2 cat#715–225-150 RRID: AB_2340826; all Jackson ImmunoResearch Laboratories) for 1.5 h. Post-incubation, slides were washed 2 x PBS, 3 × 5 min with blocking solution and 2 x PBS. All slides were mounted to coverslips using PBS/Glycerol (1:1).
4
LPS-Induced Cytoskeletal Dynamics in Cells
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LPS from Escherichia coli 055:B5 was obtained from Sigma (St. Louis, MO, USA), and UFH was obtained from Shanghai No.1 Biochemical & Pharmaceutical Co. (China). Rabbit polyclonal α-tubulin, GEF-H1, p-MYPT1, p38 MAPK, and p-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal acetylated-α-tubulin and the p38/MAPK inhibitor SB203580 were obtained from Abcam (Cambridge, MA, USA). Nocodazole was purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal antibody against GAPDH was from Invitrogen (Carlsbad, CA, USA). Tetraethyl rhodamine isothiocyanate (TRITC)-phalloidin (Solarbio, China), donkey anti-mouse Cy2 (Jackson, USA), and donkey anti-rabbit Alexa Fluor® 488 (Abcam, USA) were used for immunofluorescence.
5
Immunohistochemical Analysis of Pancreatic Islets
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Tissue samples were fixed in 10% formalin solution, and paraffin blocks were cut into 5-mm sections. For immunofluorescence staining, sections were prepared and stained using guinea pig anti-insulin (DAKO, Carpinteria, CA) and mouse anti-glucagon (Santa Cruz Biotechnology, Dallas, Texas). Detection was performed with the secondary antibody donkey anti-guinea pig Cy3 and donkey anti-mouse Cy2 (Jackson, West Grove, PA) for 20 min at room temperature. Slides were mounted using ProLong Gold reagent with DAPI. Immunohistochemistry for CD4, CD8, CD19 (Abcam, Cambridge, MA) and CD138 (BD Biosciences, San Jose, CA), staining was carried out using the Vector ImmPRESS Polymer System, Peroxidase (HRP) Substrate After de-paraffinization, antigen retrieval was carried out by boiling the slides in tris-based, pH 9 (Vector, Burlingame, CA) for 20 min. All antibodies at optimal dilution were incubated overnight at 4°C. Slides were then incubated with anti-rabbit or Rat HRP polymer for 30 min at room temperature followed by DAB+ substrate-chromogen solution for 5 min at room temperature. Slides were counterstained with hematoxylin and mounted.
6
Immunofluorescence Staining of Neural Tissues
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Tissues for histology were fixed in 4 % paraformaldehyde (PFA), cryoprotected in 30 % sucrose and frozen in Richard-Allan™ Scientific Neg-50™ Frozen Section Medium (Thermo Fisher Scientific, Waltham, MA, USA). Sections with thickness of 5–20 µm were used for immunofluorescence staining. Following antibodies/reagent were used for immunofluorescence staining: Rabbit anti-GFAP (1:300, Agilent), rabbit anti-Ki67 (1:200, Thermo Fisher Scientific), goat Sox2 (1:200, Santa Cruz Biotechnology, Dallas, United States), goat anti-doublecortin (DCX) (1:200, Santa Cruz), mouse anti-GFP (1:200, Santa Cruz), donkey anti-rabbit Cy5 (1:200 – 1:400, Jackson ImmunoResearch, West Grove, United States), rabbit anti-goat IgG Cy3 (1:200 – 1:400, Sigma-Aldrich, St. Louis, USA), Donkey anti-mouse Cy2 (1:200 – 1:400, Jackson ImmunoResearch) and DAPI (1:3000–1:5000, Thermo Fisher Scientific).
7
Immunostaining of Cytoskeletal Proteins
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Cells were fixed in 4% paraformaldehyde
in PBS for 15 min, permeabilized with 0.1% Triton X for 10 min, and
blocked with 1% BSA in PBS for 30 min. The cells were then incubated
with primary antibody for 1 h at room temperature and overnight at
4 °C, followed by a 2 h incubation at room temperature with secondary
antibody and a 10 s incubation with DAPI for nuclear staining. Primary
antibodies were monoclonal mouse anti-α-smooth muscle actin
(α-SMA, Sigma, 1:500), polyclonal rabbit antidesmin (Abcam,
1:80), and antihuman lumican (R&D Systems, 1:400). Secondary antibodies
were cy2 donkey antimouse (1:200), cy3 donkey antirabbit (1:200),
and cy3 donkey antigoat (1:200), all from Jackson Immunoresearch Laboratories.
in PBS for 15 min, permeabilized with 0.1% Triton X for 10 min, and
blocked with 1% BSA in PBS for 30 min. The cells were then incubated
with primary antibody for 1 h at room temperature and overnight at
4 °C, followed by a 2 h incubation at room temperature with secondary
antibody and a 10 s incubation with DAPI for nuclear staining. Primary
antibodies were monoclonal mouse anti-α-smooth muscle actin
(α-SMA, Sigma, 1:500), polyclonal rabbit antidesmin (Abcam,
1:80), and antihuman lumican (R&D Systems, 1:400). Secondary antibodies
were cy2 donkey antimouse (1:200), cy3 donkey antirabbit (1:200),
and cy3 donkey antigoat (1:200), all from Jackson Immunoresearch Laboratories.
8
Immunocytochemistry of Induced Neurons
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Coverslips (Electron Microscopy Sciences #7229102) were coated with poly-L-ornithine, laminin, and Matrigel as descrbied above for routine iN cultures. On DIV21, iNs were washed once with PBS, then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Following fixation, iNs were washed with PBS for 15 minutes at room temperature then incubated in blocking buffer (2% donkey serum with 0.1% Triton X-100) for 1 h shaking at room temperature. iNs were incubated in primary antibodies diluted in blocking buffer overnight at 4 °C. Following incubation iNs were washed with PBS 3 times for 10–15 min and incubated in secondary antibodies for 1 h at room temperature. Secondaries included Cy2 Donkey anti-rabbit, Cy2 Donkey anti-mouse, Cy3-Donkey anti-mouse, Cy3-Donkey anti-rabbit, Cy5-Donkey anti-chicken 1:2000, (Jackson Immunoresearch). iNs were then washed with PBS 3 times. DAPI (1:1000) staining was performed during the second wash with PBS. Coverslips were mounted and imaged using Zeiss LSM710 confocal microscopy, 40× oil objective and acquired using Zen black software.
9
Immunostaining of DRG Neuron Development
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DRG neurons of different time of growth (from 3 to 12 DIV) were fixed for 20 min with 4% paraformaldehyde (Sigma) rinsed in PBS (0.01 M, pH 7.4) at room temperature, followed by washes with PBS, blocking in 5% BSA (Sigma) with 0.5% of triton (Sigma), and incubation with primary antibodies overnight at 4℃ in 1% BSA with 0.05% of triton. Following day, the primary antibodies were washed out by 1 × PBS and followed by incubation with Cy2, Cy3-conjungated secondary antibodies (Jackson ImmunoResearch) for 2 h at Troom in dark.
Primary antibodies: goat anti-Nav1.8 (1:50, Santa Cruz),22 (link) rabbit anti-TRPV1 (1:50, Santa Cruz),23 (link)–25 (link, link) rabbit anti-TRPM8 (1:50, Santa Cruz), rat anti-CD77 (1:10, Abcam),26 (link) mouse anti-NeuN (1:2000, Millipore),27 (link) and rabbit anti-Tuj1 (β3 Tubulin, 1:50, Santa Cruz).28 (link)Secondary antibodies: Cy2-donkey anti-rabbit, Cy2-donkey anti-mouse, Cy3-donkey anti-rat, and Cy3-donkey anti-rabbit (1:200, all from Jackson ImmunoResearch). DAPI (dilactate; 300 nM, Invitrogen) was used for counterstaining.
Primary antibodies: goat anti-Nav1.8 (1:50, Santa Cruz),22 (link) rabbit anti-TRPV1 (1:50, Santa Cruz),23 (link)–
10
Immunohistochemical Analysis of Dorsal Root Ganglia
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Samples of whole DRG were isolated from 8 - to 10-week-old animals and immediately fixed in 4% paraformaldehyde (Sigma) rinsed in PBS (0.01 M, pH 7.4) at 4℃ for three days. After the fixation, we replaced the fixative solution with cryoprotective solution of 30% sucrose (Sigma). The spinal cord and DRG were cut into 10 and 20 µm sections, respectively. The 20 µm sections were incubated with hematoxylin and eosin staining solutions, and the 10 µm were incubated with rabbit anti-TRPV1 (1:50, Santa Cruz) and mouse anti-NeuN (1:2000, Millipore) overnight at 4℃ in 1% BSA with 0.05% of Triton, then incubated with Cy2-donkey anti-mouse and Cy3-donkey anti-rabbit (1:200, from Jackson ImmunoResearch) for 2 h at Troom in dark. DAPI (dilactate; 300 nM, Invitrogen) was used for counterstaining. Images were taken by a confocal microscope.
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