Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used. Secondary antibodies, Goat Anti-Rabbit IgG–HRP conjugate (Bio Rad, #170-6515, 1:2000), Goat Anti-mouse IgG–HRP conjugate (Bio Rad, #170-6516, 1:2000), Alexa Fluor 568 goat anti Rat IgG (Invitrogen, # A-11077, 1:1000), Alexa Fluor 488 goat anti mouse IgG (Invitrogen, # A11001, 1:1000), Alexa Fluor-488-goat anti rabbit IgG (Invitrogen, #A11008), Alexa Fluor-546-goat anti mouse IgG (Invitrogen, #A11003), Alexa Fluor-546-goat anti rabbit IgG (Invitrogen, #A11010), Alexa Fluor-488-goat anti mouse IgG (Invitrogen, #A11001)were used.
Alexa fluor 568 goat anti rat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568 goat anti-rat IgG is a fluorescent secondary antibody conjugate. It is designed to bind to rat immunoglobulin G (IgG) for detection and visualization purposes in various immunoassay and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Freezing sliding microtome, by Thermo Fisher Scientific (2 mentions)
Mab367, by Merck Group (2 mentions)
D1306, by Thermo Fisher Scientific (2 mentions)
Nb600 308, by Novus Biologicals (2 mentions)
Lsm 780 or 710 confocal microscope, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Anti vegfr2, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp conjugate, by Bio-Rad (1 mentions)
Goat anti mouse igg hrp conjugate, by Bio-Rad (1 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti p erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti p csrc, by Santa Cruz Biotechnology (1 mentions)
Anti ptp1b d 4, by Santa Cruz Biotechnology (1 mentions)
Anti rab5, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
18 protocols using alexa fluor 568 goat anti rat igg
1
Protein Expression and Imaging Protocol
2
Immunostaining of IL-10 Receptor Alpha
3
Immunofluorescence Staining of Liver Tissue
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Livers were frozen in O.C.T compound and sectioned at 4 μm-thick. After fixation with ice-cold acetone for 3 min, the sections were incubated with rat anti-mouse F4/80 (eBioscience) and goat anti-mouse TNFα antibody (R&D Systems) overnight at 4 °C. Tissue sections were then washed with TBS-T and incubated for 30 min with Alexa Fluor 647 rabbit anti-goat IgG and Alexa Fluor 568 Goat anti-rat IgG (Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear staining. The tissue sections were analysed using an Olympus confocal microscope system (Olympus, Tokyo, Japan).
4
Immunofluorescent Labeling of Mouse Brain
5
Endothelial Cell Staining in Matrigel
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CD31 antibody, which was used to stain the endothelial cells in Matrigel, was purchased from Novus Biologicals, Inc. (Littleton, CO). Secondary antibodies of Alexa Fluor 568 goat anti-rat IgG was purchased from Invitrogen (Carlsbad, CA). Growth Factor Reduced Matrigel™ Matrix (Phenol Red-free) and Cell Recovery Solution were purchased from BD Pharmingen (San Diego, CA). Mouse VEGF was purchased from R&D Systems (Minneapolis, MN). Oligofectamine and Opti-MEM were purchased from Life Technologies (Grand Island, NY). SiRNA targeting EGFL7 and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
6
Immunofluorescent Labeling of Mouse Brain
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Mice were anesthetized with sodium pentobarbital (40 mg/kg) before transcardial perfusion with PBS, immediately followed by 4% paraformaldehyde (PFA) in PBS, pH 7.4. Brains were post fixed overnight at 4°C before sequential 24 h incubations in PBS with 10%, 20%, and 30% sucrose. Brains were sectioned coronally at 40 μm using a freezing sliding microtome (Thermo Scientific). Sections were stored at 4°C in a cryopreservative solution (45% PBS, 30% ethylene glycol, 25% glycerol, by volume). For immunostaining, brain sections were rinsed several times with PBS before blocking with 5% normal goat serum and 0.02% Triton X-100 in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies, including rat anti-M2 AChR antibody (1:500, MAB367, Millipore) and rabbit GFP antibody (1:500, NB600-308, Novus), diluted in normal goat serum at 4°C overnight, and then rinsed several times with 0.02% Triton X-100 in PBS. Tissues were then incubated with secondary antibodies, including Alexa Fluor-488 goat anti-rabbit IgG (1:500, #A11008, Invitrogen) and Alexa Fluor-568 goat anti-rat IgG (1:500, #A11077, Invitrogen) for 1 hour. 4’,6-diamidino-2-phenylindole (DAPI, 7mg/mL, D1306, Invitrogen) was added during the secondary antibody incubation for nuclear counterstaining. Images were acquired with a Zeiss LSM 780 or 710 confocal microscope.
7
Immunohistochemical Analysis of RLIM Expression
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Primary antibodies used for immunostainings were rabbit RLIM (Ostendorff et al. 2002) , Lectin PNA from Arachis hypogaea (peanut), GATA1 (Santa Cruz Biotechnology, sc265), GATA4 (Abcam, ab84593), pPKAs (Cell Signaling, # 9624), PY (Millipore, clone G10), Alexa Fluor 488-conjugated lectin peanut agglutinin (ThermoFisher, # L21409).
Secondary antibodies were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (Invitrogen, A21206), Alexa Fluor® 488 Goat Anti-mouse IgG (Invitrogen, A11029), Alexa Fluor® 568 Goat Anti-Rabbit IgG (Invitrogen, A11011), Alexa Fluor® 568 Goat Anti-rat IgG (Invitrogen, A11077), and Alexa Fluor® 568 Goat Anti-mouse IgG (Invitrogen, A11004).
Immunohistochemical (co-)staining on paraffin-embedded tissue sections were carried out as previously reported (Shin et al. 2010) . Northern blot analysis was performed on a membrane containing RNA from adult mouse tissues (Clontech). As probe, we used a 568 bp SacI fragment isolated from mouse Rlim cDNA, which was 32 P-labeled via random priming (Gibco BRL) as previously described (Ostendorff et al. 2000) .
Secondary antibodies were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (Invitrogen, A21206), Alexa Fluor® 488 Goat Anti-mouse IgG (Invitrogen, A11029), Alexa Fluor® 568 Goat Anti-Rabbit IgG (Invitrogen, A11011), Alexa Fluor® 568 Goat Anti-rat IgG (Invitrogen, A11077), and Alexa Fluor® 568 Goat Anti-mouse IgG (Invitrogen, A11004).
Immunohistochemical (co-)staining on paraffin-embedded tissue sections were carried out as previously reported (Shin et al. 2010) . Northern blot analysis was performed on a membrane containing RNA from adult mouse tissues (Clontech). As probe, we used a 568 bp SacI fragment isolated from mouse Rlim cDNA, which was 32 P-labeled via random priming (Gibco BRL) as previously described (Ostendorff et al. 2000) .
8
Immunofluorescence Staining for MERS-CoV Detection
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To identify virus-positive cells, cells on chamber slides and Transwell inserts, human intestinoids, slides of human small intestine, or mouse tissues were subjected to immunofluorescence staining, as described previously (45 (link)). Briefly, after permeabilization and blocking, cells, intestinoids, and tissues were stained with a guinea pig antiserum against MERS-CoV NP, followed with secondary antibodies including goat anti–guinea pig Alexa Fluor 488 immunoglobulin G (IgG) or goat anti–guinea pig Alexa Fluor 594 IgG (Abcam). For intestine explants and intestinoids, CK19 (YM3051, ImmunoWay), a marker of intestinal epithelial cell, was costained with an anti-NP antibody. To define the identity of the viral NP-positive cells in the tissues of infected hDPP4 mice, apart from the labeling of MERS-CoV NP, double staining was performed using a rat anti-mouse monoclonal CD68 antibody (FA-11, Abcam) and Alexa Fluor 568 goat anti-rat IgG (Life Technologies). After staining, intestinoids were whole-mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories). The whole-mounted intestinoids, chamber slides, Transwell inserts, and tissue slides were imaged using a Carl Zeiss LSM 780 or 800 confocal microscope.
9
Immunostaining of GFP-labeled Cells
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Sorted cells were collected in 200 µl of 1× PBS and seeded in a Nunc glass-bottom dish (Thermo Fisher Scientific, #150680) previously treated with 200 µl of 20 µg ml−1 fibronectin (Sigma-Aldrich, #F1141-2MG) overnight at 4°C. Cells were incubated for 3 h at 23°C, then 1× PBS was substituted with 200 µl growth medium and grown overnight at 23°C.
Cells were fixed for 5 min at room temperature with 4% formaldehyde in 1× PBS and washed once with 200 µl 1× PBS. Cells were then blocked for 30 min at room temperature in blocking solution [1% bovine serum albumin (Sigma-Aldrich, #A3294-10G), 0.1% Triton-X100 (Sigma-Aldrich, X100) in 1× PBS] and incubated for 1.5 h at room temperature with 1:100 anti-green fluorescent protein (GFP) primary antibody (Abcam, ab5450, Lot GR277059-1) in blocking solution [Venus is an improved version of GFP (Nagai et al., 2002 (link))]. Cells were washed twice for 10 min in blocking solution and incubated for 1.5 h in the dark at room temperature with 1:1000 Alexa Fluor 568 goat anti-rat IgG (Life Technologies, A11077, Lot 1512105) in the blocking solution. After three washes of 10 min in 1× PBS, the preparation was overlaid with fluorescence mounting media (DAKO/Agilent Technologies, #S3023), covered with a coverslip and sealed with nail polish.
Cells were fixed for 5 min at room temperature with 4% formaldehyde in 1× PBS and washed once with 200 µl 1× PBS. Cells were then blocked for 30 min at room temperature in blocking solution [1% bovine serum albumin (Sigma-Aldrich, #A3294-10G), 0.1% Triton-X100 (Sigma-Aldrich, X100) in 1× PBS] and incubated for 1.5 h at room temperature with 1:100 anti-green fluorescent protein (GFP) primary antibody (Abcam, ab5450, Lot GR277059-1) in blocking solution [Venus is an improved version of GFP (Nagai et al., 2002 (link))]. Cells were washed twice for 10 min in blocking solution and incubated for 1.5 h in the dark at room temperature with 1:1000 Alexa Fluor 568 goat anti-rat IgG (Life Technologies, A11077, Lot 1512105) in the blocking solution. After three washes of 10 min in 1× PBS, the preparation was overlaid with fluorescence mounting media (DAKO/Agilent Technologies, #S3023), covered with a coverslip and sealed with nail polish.
10
Multi-Staining Immunohistochemistry Imaging
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Immunohistochemical staining experiments were performed as described [16 (link),33 (link)]. mAb nc82 (1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) was used for visualization of the synaptic neuropil. Rat anti-5HT (1:100; Abcam, Cambridge, MA, USA) staining marked the serotonergic projection neurons. Anti-cleaved caspase-3 (Cell Signaling Techology, Danvers, MA, USA) is a marker for cell death. Anti-acetylated tubulin (Zymed, San Francisco, CA) staining marks axons. The following secondary antibodies were used at a concentration of 1:200: goat anti-mouse FITC, goat anti-mouse Cy3, goat anti-rabbit FITC (Jackson Immunoresearch, West Grove, PA, USA), and Alexa Fluor 568 goat anti-rat IgG (Life Technologies, Grand Island, NY, USA). Tissues were imaged with a Zeiss 710 confocal microscope using Zen software, and scanned images were analyzed with FIJI and Adobe Photoshop software.
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