Cryosections of optimum cutting temperature (OCT) embedded CD1 tissues were incubated with monoclonal anti-TSP-1 (1:200; Abcam). After overnight incubation in the primary antibody solution and blocking buffer, the samples were exposed to goat anti-mouse Alexa Fluor 568 secondary antibody (1:200; Invitrogen). Finally, the nuclei were counterstained with DAPI (Invitrogen). The samples were coverslipped with DAKO mounting media (DAKO). Scleras and retinas were imaged with a Zeiss LSM 710 Confocal Microscope (Zeiss MicroImaging, Thornwood, NY) at the peripapillary region using a 20X lens. We used two control procedures in the mouse sclera tissue when we used antibodies generated in mice against the molecules studied. First, we blocked immunoglobulin (IgG) labeling with serum, and, second, we treated some control sclera after we omitted the primary antibody as controls. These procedures demonstrated that there was no detectable non-specific labeling. Masked observers graded the amount of Thrombospondin 1 and Hint1 in the scleral sections of the control and glaucomatous tissues. Images were evaluated on a scale of 1–5; 1 was the lowest signal, and 5 was the highest. Individual and average gradings from each observer were statistically analyzed.
Alexa fluor 568 goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 568 goat anti-mouse secondary antibody is a fluorescently labeled antibody used in immunoassays to detect and visualize mouse primary antibodies. The antibody is conjugated with the Alexa Fluor 568 dye, which emits a red fluorescent signal upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 confocal microscope, by Leica (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (3 mentions)
Mouse anti occludin primary antibody, by Thermo Fisher Scientific (3 mentions)
Draq5, by Thermo Fisher Scientific (3 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (3 mentions)
Mouse anti dsred antibody, by Takara Bio (2 mentions)
Rabbit anti npy primary antibodies, by Merck Group (2 mentions)
Evos imaging system and microscope, by Thermo Fisher Scientific (2 mentions)
Immuno mount, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Axioplan 2, by Zeiss (2 mentions)
Nocodazole, by Merck Group (2 mentions)
γh2ax ser139, by Merck Group (2 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Dako mounting media, by Agilent Technologies (1 mentions)
Ici 182 780, by Bio-Techne (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
21 protocols using alexa fluor 568 goat anti mouse secondary antibody
1
Quantifying Thrombospondin 1 in Glaucoma Sclera
2
Immunochemical Analysis of Estrogen Receptor
3
Visualizing dCas9-ED Protein Expression
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HEK293 cells were fixed and stained at different time points between 3 and 9 days after transfection for the two components of the functional dCas9-ED by immunofluorescence staining. Cells were fixed with 4% PFA (Sigma-Aldrich #F8775) for 10 min in PBS with 0.5% Triton X-100 and blocked subsequently with 1% goat serum in PBS applied overnight. Mouse anti-Flag M2 monoclonal primary antibody (Sigma #F1804) and goat anti-mouse Alexa Fluor 568 secondary antibody (Invitrogen #A11004) were used to label the dCas9-ED. Successful transfection with the sgRNA plasmid was detected with rabbit anti-GFP monoclonal primary antibody (Cell Signaling Technology #2956, Danvers, MA) and goat anti-rabbit Alexa Fluor 488 (Molecular Probes #A11034, ). DNA was stained with DAPI.
4
Immunohistochemical Staining of Neural Markers
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For IHC, 4 animals were anesthetized with isoflurane and transcardially perfused with cold Ringer’s solution containing 2% lidocaine HCl (20 mg/mL) and heparin (1000 USP units/mL) followed by 4% paraformaldehyde. Brains were harvested as previously described 19 (link). Immunostaining for tdTomato was performed with a 1:200 diluted mouse anti-DsRed antibody (Clontech, Mountain View, CA). For PVALB immunostaining, we used a 1:5,000 dilution of rabbit anti-parvalbumin primary antibody (Swant Ltd., Marly, Switzerland). CCK-stained sections were incubated with rabbit anti-proCCK (a generous gift from Dr Andrea Varro; 1:1000) for 72 h at 4°C, while NPY staining was performed using rabbit anti-NPY primary antibodies (Sigma, St Louis, MO, USA; 1:1000) . For fluorescence visualization, goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 568 secondary antibodies were used (Life Technologies, Carlsbad, CA, USA) at 1:250 dilution. DAPI staining was performed using 3 min incubation in 300 nM DAPI (Sigma, St Louis, MO, USA). Rinsed brain slices where mounted on slides and cover-slipped in ImmunoMount (Fisher Scientific, Pittsburg, PA, USA). Imaging was performed using the EVOS imaging system and microscope (Life Technologies, Carlsbad, CA, USA).
5
Immunohistochemical Staining of Neural Markers
6
Immunostaining of Transgenic Zebrafish Larvae
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Phenylthiourea treated and anaesthetized EGUSG larvae at 2.5 dpf were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). To enhance permeability, larvae were incubated in 0.1% trypsin and 0.5% hyaluronidase in PBS for 1 h and 1.5 h, respectively. For section immunostaining, larvae were fixed in 4% PFA and dehydrated with methanol and then embedded for frozen cryostat sectioning. The following antibodies and concentrations were used for whole-mount and frozen section double-IHC: rabbit polyclonal anti-GFP primary antibody (Molecular Probe), 1:800; rabbit polyclonal anti-RFP (including DsRed as the antigen) primary antibody (MBL), 1:500; mouse monoclonal anti-SV2 primary antibody (Hybridoma Bank, Iowa City, IA), 1:50; Alexa Fluor 488 goat-anti-rabbit secondary antibody (Molecular Probe), 1:1000; Alexa Fluor 633 goat-anti-rabbit secondary antibody (Molecular Probe); Alexa Fluor 568 goat-anti-mouse secondary antibody (Molecular Probe), 1:1000.
7
Visualizing ZFP36L1 effects on IAV
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A549 cells were transduced with the lentiviral vector expressing HA-tagged ZFP36L1 protein for 72 h, then infected with IAV for 24 h. Cells were fixed with 4% formaldehyde and permeabilized in phosphate buffered saline (PBS) with 0.5% Triton X-100. IAV NP protein expression was detected with a rabbit anti-IAV NP antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes). The expression of HA-tagged ZFP36L1 was detected with a mouse anti-HA antibody and Alexa Fluor 568 goat anti-mouse secondary antibody (Molecular Probes). Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI; Molecular Probes).
8
Mitotic Arrest and DNA Damage Response
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C57BL/6J (p53-/-) MEFs (8 x 105) were plated in 10-cm plates. The next day cells were incubated with 100 ng/ml Nocodazole (Sigma) for 6 h and mitotic cells harvested using the shake-off method. At this point supernatant was taken for the t=0 time point. Mitotic cells (1 x 105) were then plated per well of a 12-well plate; or 7 x 104 on coverslips for immunofluorescence analysis. Asynchronous cells were plated at the same number concurrently. Supernatants were taken and coverslips fixed at 6, 16 and 22 h. Immune response was analysed by ELISA and coverslips were pre-extracted on ice using 0.5% Triton-X100 in PBS for 5 min, cells were then fixed with 4% PFA for 15 min at room temperature (RT). After blocking for 30 min with 3% BSA at RT, γH2AX Ser139 (05-636 Millipore) was added for 2 hr at RT. Alexa Fluor 568 goat anti-mouse secondary antibody (Life technologies) was then applied and incubated for 1 h at RT. Coverslips were mounted using Vectashield antifade mounting medium with DAPI (Vector laboratories) and imaged at RT using a Coolsnap HQ CCD camera (Photometrics) and a Zeiss Axioplan II fluorescence microscope with x40 and x63 Plan-neofluar objectives and acquired using micromanager (http://open-imaging.com/ ) n≥100 cells per condition (n=1).
9
Mitotic Arrest and DNA Damage Response
10
Immunolocalization of R. parkeri in Ticks
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The infection status of R. parkeri infected (Rp+) ticks was confirmed by immunolocalization. This was done using unfed and partially fed salivary glands and ovarian tissues. Dissected tissues were fixed in 4% PFA and 4% sucrose diluted in 1X PBS and kept at 4°C until needed. Fixed samples were washed three times in 1X PBS prior to permeabilization. Samples were permeabilized in 0.25% Triton X-100 in PBS for 30 min followed by blocking in 2% BSA in PBS for an additional 1 h. Tissues were incubated overnight at 4°C with anti-Rickettsia antibody (M14-13, 1:500, kindly provided by Dr. Ted Hackstadt) that recognizes R. parkeri in 1X PBS containing 2% BSA. This was followed by incubation in the dark with Alexa-Fluor 568 goat anti-mouse secondary antibody (1,500, Invitrogen, Thermo Fisher Scientific, Eugene, Oregon, United States) in 1X PBS containing 2% BSA. Samples were washed three times to remove unbound antibodies and mounted on glass slides using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, United States).
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