For immunofluorescence analysis, rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and transcardially perfused them with 100 ml of heparinized normal saline, followed by 500 ml freshly prepared fixative containing 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The TG sections were permeabilized with 50% ethanol for 30 min, blocked with 10% normal donkey serum (NDS) for 30 min, and incubated overnight in the primary antibodies [goat polyclonal antibody to GRP78 (sc1050, 1:200, Santa Cruz Biothechnology), rabbit monoclonal antibody to p-eIF2α (#3597, 1:500, Cell Signaling) and the neuronal marker, microtubule associated protein (MAP2; #ab11267, 1:1,000, Abcam)]. TG sections were washed with 0.01 M phosphate buffered saline (PBS, pH 7.4) and incubated with 2% NDS for 30 min, and incubated with secondary antibodies [Cy3-conjugated-donkey anti-goat antibody, Cy3-conjugated-donkey anti-rabbit antibody and FITC-conjugated-donkey anti-mouse antibody (1:200 in PBS; Jackson Immunoresearch)] for 3 h. Finally, sections were rinsed with PBS, mounted on slides, and coverslipped with Vectashield (Vector, Burlingame, CA) solution. Microscopic observation was performed with an Exi digital camera (Q-Imaging Inc, Surrey, CA) attached to a Zeiss Axioplan 2 conventional fluorescence microscope (Carl Zeiss Inc, Jena, Germany).
Cy3 conjugated donkey anti goat antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated donkey anti-goat antibody is a secondary antibody that is conjugated to the fluorescent dye Cy3. It is designed to detect and bind to goat primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab11267, by Abcam (1 mentions)
Fitc conjugated donkey anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Cy3 conjugated donkey anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti gfap antibody, by Proteintech (1 mentions)
Mouse anti neun antibody, by Merck Group (1 mentions)
Mouse anti ha antibody, by BioLegend (1 mentions)
Goat anti olig2 antibody, by R&D Systems (1 mentions)
Alexa fluor 488 anti ha 11 epitope tag antibody, by BioLegend (1 mentions)
Cy3 conjugated goat anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Goat polyclonal antibody against iba 1, by Abcam (1 mentions)
Fluorescein isothiocyanate fitc albumin, by Merck Group (1 mentions)
Fitc albumin, by Abcam (1 mentions)
Af2125, by R&D Systems (1 mentions)
Vectashield with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
4 protocols using cy3 conjugated donkey anti goat antibody
1
Immunofluorescence Analysis of Trigeminal Ganglia
2
Immunofluorescence Assay for Detecting Viral Proteins
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The following antibodies were used: mouse anti-HA antibody (1:500, BioLegend), rabbit anti-Giantin antibody (1: 1000, Biolegend), rabbit anti-PDI antibody (1:50, CST), goat anti-Olig2 antibody (1:200, R&D), rabbit anti-GFAP antibody (1:200, Proteintech), rabbit anti-IBA1 antibody (1:250, Invitrogen), mouse anti-Neu-N antibody (1:100, Millipore), rabbit anti-HCoV-OC43-N antibody, Cy3-conjugated donkey anti-rabbit antibody (1:500, Jackson), Alexa Fluor 488-conjugated donkey anti-mouse antibody (1:500, Jackson), Alexa Fluor 555-conjugated donkey anti-mouse antibody (1:500, Jackson), Alexa Fluor 488 anti-HA.11 Epitope Tag Antibody (1:500, Biolegend), Cy3-conjugated donkey anti-goat antibody (1:500, Jackson).
17CL-1 Cells infected with the virus (MOI = 0.1) were fixed using 4% PFA in PBS, then the cells permeabilized with 0.2% Triton X-100 in PBS. Cells were incubated with primary antibody at RT for 1 h, and then incubated with second antibody at RT for 1 h, the nucleus was stained with DAPI. After three times wash with PBS, the coverslips were mounted on glass slides. Cells were examined by confocal microscopy (ZEISS).
17CL-1 Cells infected with the virus (MOI = 0.1) were fixed using 4% PFA in PBS, then the cells permeabilized with 0.2% Triton X-100 in PBS. Cells were incubated with primary antibody at RT for 1 h, and then incubated with second antibody at RT for 1 h, the nucleus was stained with DAPI. After three times wash with PBS, the coverslips were mounted on glass slides. Cells were examined by confocal microscopy (ZEISS).
3
Assessing Blood-Brain Barrier Integrity
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To detect BBB leakage in 3-NP-injected rats, experimental rats (n = 3 animals) were deeply anesthetized on days 3 and 7 after 3-NP injection and intravenously administered fluorescein isothiocyanate (FITC)-albumin (2 mg diluted in 0.1 ml saline; Sigma-Aldrich) via the tail vein. One hour after injection, animals were deeply anesthetized and perfused transcardially with fixative, as described above. Coronal cryostat sections (25-μm-thick) from animals injected with FITC-albumin were incubated overnight at 4°C with a mixture of rabbit polyclonal antibody against GRP78 (1:2000; Abcam) and one of following antibodies: goat polyclonal antibody against Iba1 (1:500; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad), or mouse monoclonal antibodies against GFAP (1:700; Millipore). This was followed by 2-h incubation with appropriate secondary antibodies as follows: Cy3-conjugated donkey anti-goat antibody (1:2000; Jackson ImmunoResearch), Cy3-conjugated goat anti-mouse antibody (1:2000; Jackson ImmunoResearch), or Alexa Fluor 647-tagged donkey anti-rabbit antibody (1:300; Thermo Fisher). Counterstaining of cell nuclei was carried out using DAPI for 10 min.
4
Corneal Neovascularization Visualization Protocol
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Corneas were excised on day 14 after suture-induced CNV and washed with phosphate-buffered saline. The corneal epithelium was removed after incubation with 20 mM EDTA for 60 min at 37 °C, fixed in acetone for 15 min at 20–22 °C, and blocked in 2% bovine serum albumin for 60 min. Corneas were then double-stained for CD31 and lymphatic vessel endothelial hyaluronan receptor LYVE-1 as described previously by using overnight incubation with goat anti-mouse CD31 FITC (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-mouse LYVE-1 (1:400; AF2125, R&D Systems, MN, USA)41 (link),54 (link). A Cy3-conjugated donkey anti-goat antibody (1:2,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was then added as the secondary antibody and incubated for another 2 h. Stained whole mount corneas were mounted in Vectashield with 4,6-diamidino-2-phenylindole (Vector Laboratories Inc., Burlingame, CA, USA). Images of stained whole mount corneas were taken under a fluorescence microscope (BZ-X71000, KEYENCE, Osaka, Japan). The fractions of total corneal areas covered by blood and lymphatic vessels were then calculated by using ImageJ (National Institutes of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/index.html) 41 (link),55 (link).
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