HIF-1α/NeuN double immunofluorescence analyses were performed in the Sham, CON, and EA groups. Monoclonal mouse anti-HIF-1α (1:200, Novus Biologicals) and polyclonal rabbit anti-NeuN (1:1000, Millipore) were used. HIF-1α/Notch1 NICD double immunofluorescence analyses of the CON and EA groups were then performed. The secondary antibody used was FITC-labeled goat anti-mouse IgG (1:2000; Molecular Probes, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:1 000; Molecular Probes, USA). Finally, sections were observed, and images were captured using an Olympus BX-60 fluorescence microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) with software named QCapture Pro.
Alexa fluor 594 conjugated anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with the Alexa Fluor 594 fluorescent dye, which can be detected using appropriate fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (23 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 594 conjugated anti mouse igg, by Thermo Fisher Scientific (5 mentions)
Vectashield, by Vector Laboratories (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Lsm800 confocal microscope, by Zeiss (4 mentions)
Axioplan 2 microscope, by Zeiss (3 mentions)
Alexa fluor 488 conjugated anti rat igg, by Thermo Fisher Scientific (3 mentions)
Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Fitc labeled goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Bx60 fluorescence microscope, by Olympus (2 mentions)
Bz 9000 fluorescence microscope, by Keyence (2 mentions)
Hybrid cell count software, by Keyence (2 mentions)
A11120, by Thermo Fisher Scientific (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Rat monoclonal anti ha 3f10, by Roche (2 mentions)
Cangetsignal immunostain a, by Toyobo (2 mentions)
Alexa fluor 488 conjugated anti rat igg, by Cell Signaling Technology (2 mentions)
64 protocols using alexa fluor 594 conjugated anti rabbit igg
1
Neuronal Hypoxia Signaling Pathway
2
Immunofluorescence Analysis of Macrophage Markers
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Cells were grown on coverslips overnight. Then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS for 15 min in room temperature. Then aspirate fixative, rinse three times in 1 × PBS for 5 min each. After treating with 0.5% Triton-X100 (ThermoFisher, USA) diluted in warm PBS and blocking specimen in blocking buffer (5% Bovine Serum Albumin diluted in warm PBS, BioFroxx, Guangzhou, China) for 60 min in room temperature, primary antibodies were applied for 4 C overnight. AlexaFluor488- conjugated anti-mouse IgG (1:1000, Molecular Probes, A11029), AlexaFluor488-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11034) or AlexaFluor594-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11037) secondary antibodies were used in room-temperature for 1 h. Nuclei were counterstained with DAPI (1 μg/ml, Molecular Probes, D1306). Protein subcellular localization was analyzed under a Zeiss 510 META or Leica TCS-SP2 confocal laser scanning microscope. The concentration of primary antibody was rabbit anti-CD68 (1:200, Abcam, ab125212), rabbit anti-CD163 (1:200, Proteintech, 16,646–1-AP), rabbit anti-CD204 (1:200, Abcam, ab123946), rabbit anti-CD206 (1:200, Abcam, ab64693), mouse anti-Arg1 (1:200, Abcam, ab239731), rabbit anti-Mrc1 (1:200, Abcam, ab64693), rabbit anti-Fizz1 (1:200, Abcam, ab39626).
3
Immunofluorescence Analysis of Ischemic Penumbra
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First, PirB/NeuN double immunofluorescence analyses in the ischemic penumbra of rats were performed on the Sham, MCAO and MCA + EA groups at 28 d after ischemia/reperfusion. Rabbit monoclonal anti-PirB antibody (1:100, Abcam, Cambridge, England) and mouse monoclonal anti-NeuN antibody (1:500, Millipore, Temecula, CA, USA) were used. The secondary antibodies were FITC-labeled goat anti-mouse IgG (1:2000, Molecular Probes, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:1000, Molecular Probes). Alternatively, immunofluorescence staining for NF200 in the ischemic penumbra of rats was performed on the Sham, MCAO and MCAO + EA groups at 28 d after ischemia/reperfusion. Staining was performed using mouse monoclonal anti-NF200 antibody (1:500, Abcam) FITC-labeled goat anti-mouse IgG secondary antibody (1:2000, Molecular Probes). Finally, the sections were examined and images were captured using an Olympus BX-60 fluorescence microscope (Olympus Corp., Shinjuku, Tokyo, Japan).
4
Immunofluorescence and Proximity Ligation Assay
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Cells were cultured on 15-mm coverslips and indicated plasmids were transfected. Cells on coverslips were washed with PBS twice and fixed with PBS containing 3% paraformaldehyde, 2% sucrose, 0.5% Triton-X-100, chilled on ice for 30 min, and then permeabilized with 0.5% Triton X-100/PBS for 5 min. After blocking with 2% BSA/PBS, cells were stained with the indicated primary antibody diluted in 2% BSA/PBS for 1 h at RT. The secondary antibodies used were Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 594-conjugated anti-rabbit IgG (Molecular Probes). PLA was performed with reagents from DuoLink Biosciences in accordance with the manufacturer's instructions. Images were captured using a BZ-9000 fluorescence microscope (Keyence). Quantification of the PLA signal dots and FANCD2 or RPA foci was determined using Hybrid cell count software (Keyence).
5
Visualizing Germline Granules via CGH-1 Immunostaining
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To visualize germline granules, immunostaining against CGH-1 was performed as previously reported (Navarro et al. 2001 (link)). Briefly the gonads of 1-d-old animals were dissected, freeze-cracked, fixed in cold methanol for 1 min, and then in 3.3% paraformaldehyde for 30 min. For coimmunostaining with anti-CGH-1 and anti-GFP, the samples were fixed for only 18 min. Then, the samples were blocked with 30% normal goat serum (NGS; Sigma-Aldrich, St. Louis, MO) in PBT for 30 min. Primary antibody incubation was performed overnight at 4° with rat anti-CGH-1 (1:25; Navarro et al. 2001 (link)). Coimmunostaining used rabbit anti-CGH-1 (1:1000; Boag et al. 2005 (link)) and mouse anti-GFP (1:5000; A11120 from Molecular Probes, Eugene, OR). Secondary antibody incubations were performed for 1.5 hr at room temperature with Cy3-conjugated donkey anti-rat IgG (1:100; H+L; 112165003, Jackson ImmunoResearch, West Grove, PA), or with Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG (1:100; H+L; A11001, Molecular Probes, Eugene, OR). To detect DNA, 1 ng/μl 4′9,6-diamidino-2-phenylindole (DAPI) was used. Vectashield (Vector laboratories, Burlingame, CA) was added to avoid photo bleaching before sealing the sample.
6
Apoptosis Quantification by Immunofluorescence and Flow Cytometry
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After 24 h of treatment with DMSO or KCH-1521, the cells were fixed with 2% PFA and blocked with 5% NGS. Briefly, the cells were incubated with cleaved caspase-3 (#9661, Cell Signaling Technology, Danvers, MA, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG (A11012, Molecular Probes). Nuclei were stained with DAPI, and immunofluorescence images were obtained using the TE-FM Epi-fluorescence system attached to an inverted microscope.
Annexin V and PI staining was performed using the FITC Annexin V apoptosis detection kit I (556547) according to the manufacturer’s instructions, and then 3 × 104 cells were analyzed using a flow cytometer (FACS Calibur) and Cell Quest Pro software (all from BD). Annexin V-negative and PI-negative cells were identified as viable, Annexin V-positive and PI-negative cells as early apoptotic, Annexin V-positive and PI-positive cells as late apoptotic, and Annexin V-negative and PI-positive cells as necrotic cells.
Annexin V and PI staining was performed using the FITC Annexin V apoptosis detection kit I (556547) according to the manufacturer’s instructions, and then 3 × 104 cells were analyzed using a flow cytometer (FACS Calibur) and Cell Quest Pro software (all from BD). Annexin V-negative and PI-negative cells were identified as viable, Annexin V-positive and PI-negative cells as early apoptotic, Annexin V-positive and PI-positive cells as late apoptotic, and Annexin V-negative and PI-positive cells as necrotic cells.
7
Evaluating Host Myelination in Rat Brain
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In order to confirm the integrity of host myelin at PND 45, the rat brain was perfusion-fixed with 10% paraformaldehyde solution and postfixed in the same solution for 48 h, followed by cryoprotection in 30% sucrose for 72 h. Paraffin-embedded sections were stained with Luxol fast blue (LFB) for examination of myelins. Coronal cryosections in 30 μm thickness were prepared and processed for immunostaining for myelin basic protein (MBP). Brain sections were incubated with primary antibody specific for MBP (1 : 200; rabbit polyclonal, Chemicon, Temecula, CA, USA) overnight at 4°C, followed by Alexa Fluor 594-conjugated anti-rabbit IgG (1 : 1,000; Molecular Probes, Eugene, OR, USA) for 2 h at room temperature [20 (link)]. All samples were evaluated immediately after staining and photographed with a laser-scanning confocal microscope (LSM710; Zeiss, New York, NY, USA). In order to quantify the immunoreactivity of host MBP, the photographs were analyzed with a Digital Image Analyzer (Image Inside; Focus, Seoul, Korea) for the red intensity, and expressed as a % of the control (normal) group.
8
Immortalized NSC Line F3 Characterization
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An immortalized NSC line, HB1.F3 (F3), was established from primary cultures of a 15-week gestational human fetal brain by infecting primary cultured NSC with a retroviral vector encoding v-myc oncogene [15 (link), 16 (link)]. F3.ChAT cells were prepared by infecting a human F3 NSC line with a retroviral vector encoding human ChAT gene as described previously [1 (link), 13 (link), 14 (link)]. F3.ChAT cells were plated on poly-l -lysine-coated Aclar plastic coverslips and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 5 min at room temperature (RT). Fixed cultures were incubated with primary antibody specific for human nestin (1 : 500, mouse monoclonal, Chemicon, Temecula, CA, USA), or human ChAT (1 : 200, mouse monoclonal, Chemicon) for 24 hr at 4°C, followed by Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 594-conjugated anti-rabbit IgG (1 : 1,000; Molecular Probes, Eugene, OR, USA) for 1 hr at RT. Cells were observed under an Olympus laser-scanning confocal microscope (LSM710; Zeiss, New York, NY, USA).
9
Visualizing Germline Stress Granules
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To visualize germline SGs, immunostaining against CGH-1 and GFP was performed as previously reported by (Huelgas-Morales et al., 2016 (link)) with some modifications. The gonads of one-day-old animals were dissected, freeze cracked, fixed in cold methanol for 1 min, and then treated with 3.3% paraformaldehyde for 15 min. The samples were washed twice with PBT, and were then blocked in PBT containing 30% of normal goat serum (NGS; Sigma-Aldrich, St. Louis, MO) for 30 min. Primary antibody incubation was performed overnight at 4°C with rabbit anti-CGH-1 (1:1000) (Boag et al., 2005 (link)) and mouse anti-GFP (1:50) (A11120 from Molecular Probes, Eugene, OR). Secondary antibody incubations were performed for 2 h at room temperature with Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG (1:100; H + L; A11001, Molecular Probes, Eugene, OR). To detect DNA, 1 ng/ul 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used. Vectashield (Vector laboratories, Burlingame, CA) was added to avoid photo bleaching before sealing the sample.
10
Immunofluorescent Detection of ISAV in Salmon
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Immunofluorescent antibody test (IFAT) for ISAV was performed as previously described [17 (link)] on frozen gills from farm IV. Briefly, sections of frozen gills were fixed in 80% cold acetone for 10 min. A polyclonal rabbit antibody to recombinant ISAV NP [16 (link)], or a monoclonal antibody to ISAV HE [17 (link)] was used for virus protein detection. Alexa Fluor® 594 conjugated anti rabbit IgG (Molecular Probes) and Alexa Fluor® 488 conjugated anti mouse IgG was used for detection of bound antibody, and sections were mounted in SlowFade® Gold (Molecular Probes).
Double immunofluorescent staining was performed with antibody to ISAV HE and monoclonal antibody to either Atlantic salmon endothelial cells [18 (link)] or polyclonal antibody to cytokeratin (AE1/AE3 (Sigma)) detecting endothelial and epithelial cells respectively as previously described [3 , 18 (link)]. Alexa Fluor® secondary antibodies (Molecular Probes) were used for detection of bound antibodies, as described above.
Double immunofluorescent staining was performed with antibody to ISAV HE and monoclonal antibody to either Atlantic salmon endothelial cells [18 (link)] or polyclonal antibody to cytokeratin (AE1/AE3 (Sigma)) detecting endothelial and epithelial cells respectively as previously described [3 , 18 (link)]. Alexa Fluor® secondary antibodies (Molecular Probes) were used for detection of bound antibodies, as described above.
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