FG-oocytes and oocytes in the growth phase (50–70 μm in diameter) were fixed as described above and incubated with rabbit monoclonal anti tri-methyl-histone H3 (Lys4) antibody (#9751, Cell Signaling Technology, New England Biolabs, Whitby, ON) (1:200) at 4°C overnight. After three washes, oocytes were incubated with goat-anti-rabbit IgG-FITC (Jackson ImmunoResearch, West Grove, PA) (1:500) at room temperature for 1 h. After three washes, the oocytes were mounted in Prolong Antifade Mounting Medium containing DAPI on Plus-coated histology slides. Images were captured and examined under a confocal microscope. Fluorescence intensity was measured in individual oocytes by ZEN software. For the oocytes in the growth phase, the mean intensity of the XX oocytes of 60–70 μm in diameter was set as 1.0 for calculating the relative intensity in all oocytes in every experiment. For FG-oocytes, the mean fluorescence intensity in the surrounded-nucleolus (SN)-type XX oocytes was set as 1.0.
Goat anti rabbit igg fitc
Manufactured by Jackson ImmunoResearch
Sourced in United States
Goat anti-rabbit IgG-FITC is a secondary antibody conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate). It is designed to detect and bind to rabbit immunoglobulin G (IgG) antibodies in various immunoassay and immunofluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab129372, by Abcam (4 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Gtx109242, by GeneTex (2 mentions)
Ab16911, by Abcam (2 mentions)
Alexa fluor 594 goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
X2 emccd, by Leica (1 mentions)
Csu x1, by Hamamatsu Photonics (1 mentions)
Acti stain 670 phalloidin, by Cytoskeleton (1 mentions)
Rabbit anti psmad1 5 9, by Cell Signaling Technology (1 mentions)
Dmi6000, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Goat anti mouse igg tritc, by Abcam (1 mentions)
Anti psd95, by Merck Group (1 mentions)
Anti neun, by Wuhan Servicebio Technology (1 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (1 mentions)
Fv300, by Olympus (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
12 protocols using goat anti rabbit igg fitc
1
Histone H3 Methylation Profiling in Oocytes
2
Immunofluorescence Analysis of Activated Oocytes
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The MII-oocytes after activation for 0, 1 and 2 h were fixed as described above, and incubated with rabbit polyclonal anti-ORC4L antibody (AbCam) (1:200) and mouse monoclonal anti-ARP2 antibody (AbCam) (1:200) at room temperature for 1 h. After three washes, oocytes were incubated with goat-anti-mouse IgG-AlexaFluor647 (Invitrogen, Life Technologies) (1:500), goat-anti-rabbit IgG-FITC (Jackson ImmunoResearch) (1:500), and Rhodamine-Phalloidin-TRITC (5 IU/ml) at room temperature for 1 h. After three washes, oocytes were transferred into Prolong Antifade Mounting Medium containing DAPI on Plus-coated histology slides. Fluorescence signals were examined under the confocal laser-scanning microscope.
3
Visualizing Microtubule Dynamics in Cells
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Cells (1 × 105/ml) were seeded in 24-well culture plates and treated with DMSO, TBPT (2 μM) or colchicine (1 μM) on the next day for 24 h. The cells were washed in PBS and then fixed in paraformaldehyde (4% v/v) for 10 min. After the cells were washed three times in PBS, they were blocked with 5% (w/v) BSA for 30 min at RT. Then, α-tubulin antibody (CST, Beverly, MA, USA) was incubated with cells in a humid chamber overnight at 4 °C. After the cells were washed three times in PBS, they were incubated with goat anti-rabbit IgG-FITC (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37 °C. Then, the cells were mounted with mounting medium containing DAPI (Vector Lab, Peterborough, UK) after they were washed three times in PBS. Finally, the cells were observed and imaged using a fluorescence microscope (Olympus IX71, Tokyo, Japan).
4
Immunofluorescence Staining Protocol in Microfluidics
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For immunofluorescence, all steps were performed within the microfluidic device at room temperature, unless otherwise stated. Cells were permeabilized with 0.1% (v/v) Triton X-100 (Cat: T8787, Sigma-Aldrich) in PBS (PBST) for 15 min and incubated with 10% (v/v) goat serum (Cat: G9023, Sigma Aldrich) in PBST for 30 min. Cells were incubated overnight at 4°C with rabbit anti-pSMAD1/5/9 (1:600 dilution, Cat: 13820, Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies goat anti-rabbit IgG-FITC (Cat: 111-095-045) or goat anti-rabbit IgG-Rhodamine Red™-X (Cat: 111-295-045) (Jackson ImmunoResearch Laboratories, Westgrove, PA, USA) were diluted 1:500 in PBST and applied for 1 h. Cells were then washed with PBST and simultaneously stained for 10 min for nuclei (DAPI) and, when needed, actin filaments (Acti-stain 670 phalloidin, 1:600 dilution; Cat: SKU PHDN1, Cytoskeleton, Denver, CO, USA). Imaging was performed using a spinning disk confocal microscope (Yokogawa CSU-X1, Hamamatsu X2 EMCCD mounted on a Leica DMI-6000).
5
Immunostaining of Brain Tissue
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The brain sections were incubated with anti-IBA-1 (1:100, cat.no.GB113502, Servicebio, Wuhan, China), anti-NeuN (1:200, cat. no.MAB377, Millipore, MA, USA), anti-PSD95 (1:200, cat. no.GB11277, Servicebio), anti-Caspase 3 (1:400, cat.no.GB11532, Servicebio), and anti-PCNA (1:400, cat.no.13110, Cell Signaling Technology, Danvers, MA,USA) primary antibody at 4 °C overnight. Subsequently, the sections were rinsed in PBS and incubated with goat anti-mouse IgG-TRITC (1:200, cat. no.ab6786, Abcam, Cambridge, MA, USA) and goat anti-rabbit IgG-FITC (1:200, Jackson Immunoresearch Laboratories, West Grove, PA, USA) secondary antibodies at room temperature for 1 h, followed by nuclei staining with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, cat.no.FMS-FZ011-050, Fcmacs, Nanjing, China). Finally, the slides were visualized using a Nikon Eclipse TiU fluorescence microscope equipped with a digital camera (FV300, Olympus, Japan).
6
Immunolocalization of CsSUS4 Protein
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Immunolocalization of CsSUS4 was performed as revealed in Sui et al. (2017) and Li et al. (2021) . Briefly, sample pretreatment including fixation, dehydration, embedding, and section cutting was done using the same method as for in situ hybridization. The sections were then blocked and incubated with a secondary antibody [goat anti-rabbit IgG-FITC, Jackson, USA, diluted 1:200] after incubation with CsSUS4 (diluted 1:200) primary antibody. Samples were incubated with 0.1% (w/v) aniline blue for 20 min in dark conditions after immunolabeling. Images were obtained under an Olympus fluoview FV1000 confocal laser scanning microscope with visualization at excitation/emission wavelength of 488/510 nm.
7
Quantifying Macrophage Polarization Markers
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Macrophages seeded on tissue arrays in M1 or M2 polarizing cytokines as described above were evaluated for expression of the M2 and M1 markers Arginase-1 and iNOS. After 24 hours, macrophages were fixed for 10 min in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in TBS for 10 min. Nonspecific protein interactions were blocked with 1% bovine serum albumin, 2% horse serum in 0.05% Tween-20. Tissue microarrays were then incubated with primary antibodies against Arginase-1 (rabbit polyclonal diluted 1:100, GTX109242, GeneTex) and iNOS (mouse monoclonal [4E5] diluted 1:300, ab129372, abcam) diluted in 1% bovine serum albumin in TBS overnight at 4°C. Arrays were washed and probed with FITC goat anti-rabbit IgG (1:250, Jackson Immuno) and Alexa Fluor-568 goat anti-mouse IgG (1:250, Invitrogen) secondary antibodies diluted in 1% bovine serum albumin for 1 hour. ECM autofluorescence was then blocked by treatment with 0.1% Sudan Black B in 70% ethanol for 20 min. Arrays were washed, coverslipped, and imaged. Variations in background autofluorescence were corrected by subtracting the mean background intensity of acellular regions of each tissue array spot using ImageJ software.
8
Immune Response to ECM Implantation
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Animal experiments were conducted in accordance with guidelines set by the Johns Hopkins University Animal Care and Use Committee. Bone and collagen tissue ECM particulate was hydrated with saline (100 mg dry wt/0.2 ml) and injected subcutaneously on the dorsum of 6–8 week old female C57BL/6 mice. Animals were sacrificed after 1 week, implants explanted, fixed in formalin, and embedded in paraffin for sectioning. Sections were then deparaffinized, rehydrated, and immunolabeled for the pan-macrophage marker F4/80 and the M1 marker iNOS. Antigen retrieval was conducted in citrate (10 mM, pH 6) for 30 min in a vegetable steamer, and rinsed with TBS in 0.05% Tween-20. Sections were blocked with 1% bovine serum albumin, 2% goat serum, and 0.05% Tween-20 for 1 hour. Sections were then incubated with primary antibodies against F4/80 (rat monoclonal [BM8] diluted 1:100, ab16911, abcam) and iNOS (mouse monoclonal [4E5] diluted 1:200, ab129372, abcam) overnight at 4°C in blocking solution. Sections were washed and probed with FITC goat anti-rabbit IgG (1:250, Jackson Immuno) and Alexa Fluor-594 goat anti-rat IgG (1:250, Invitrogen) secondary antibodies diluted in blocking solution for 1.5 hours. ECM autofluorescence was then blocked by treatment with 0.1% Sudan Black B in 70% ethanol for 20 min. Sections were counterstained with DAPI for 5 min, washed, coverslipped, and imaged.
9
Quantifying NF-κB Translocation in Salmonella-Challenged Leukocytes
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Peritoneal leukocytes were harvested by lavage at 0 (saline alone), 4, and 12 h post IP Salmonella challenge. Leukocytes were fixed in 1% formaldehyde. Fixed cells were washed twice in permeabilization buffer (PBS, 2% FCS and saponin 0.1%) followed by incubation with unlabeled rabbit IgG anti-mouse NF-κB p65 (Santa Cruz Biotechnologies, Dallas, TX) for 30 min on ice and 20 min at room temperature. FITC goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc., Baltimore, MD) were added and incubated for 20 min. Draq5 was used for nuclear staining. Imaging Flow Cytometry was used to record 10,000 events (ImageStream®X Mk II, MilliporeSigma). Ideas software was used to measure co-localization values of NF-κB and Draq5. Events with high NF-κB and Draq5 co-localization values indicate high NF-κB translocation. In contrast, events with low co-localization values indicate low NF-κB translocation.
10
Quantifying Macrophage Polarization Markers
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