Following the electrophysiological experiments, slices were fixed in 4% PFA in 0.01 M PBS at least overnight. After rinsing with PBS, slices were incubated in 0.01 M PBS blocking solution containing 2% Triton X-100 (Sigma-Aldrich) and 5% normal donkey serum (NDS) for 1 hour at RT. To visualize biocytin-filled cells, slices were next incubated with a blocking solution containing 1% Triton X-100, 5% NDS, chicken anti-GFP antibody (1:1000; Aves, catalog no. GFP-1020), and Alexa Fluor 647–conjugated streptavidin overnight on shaker at 4°C. The following day, slices were rinsed with 0.01 M PBS and incubated with Alexa Fluor 488–conjugated donkey–anti-chicken (1:500; Jackson ImmunoResearch, catalog no. 705-745-155) for 2 hours at RT. After rinsing, slices were mounted with Aqua-Poly/Mount (Polysciences, 18606-20). A subset of slices was costained with mouse anti-NeuN (1:300; Millipore, MAB377) and Alexa Fluor 568–conjugated donkey anti-mouse (1:300; Thermo Fisher Scientific, A10037) antibodies to confirm neuronal identity of the biocytin-filled cells. Fluorescence images were taken with a confocal microscope (LSM 800, Zeiss; 20× objective lens) as z-stack (2-μm interval) tiled images to cover the extent of the cell’s dendritic and axonal processes.
Alexa fluor 568 conjugated donkey anti mouse
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568-conjugated donkey anti-mouse is a secondary antibody reagent. It is designed to detect and visualize mouse primary antibodies in various immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated donkey anti rabbit, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647 conjugated donkey anti mouse, by Thermo Fisher Scientific (3 mentions)
Lavision ultramicroscope 2 light sheet, by Miltenyi Biotec (2 mentions)
Alexa fluor 568 conjugated donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
A21271, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti chicken, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated donkey anti chicken, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Nb300 213, by Novus Biologicals (1 mentions)
Dp30bw camera, by Olympus (1 mentions)
Ab9260, by Merck Group (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
T8660, by Merck Group (1 mentions)
Mab1976, by Merck Group (1 mentions)
Mab1969, by Merck Group (1 mentions)
Af8047, by R&D Systems (1 mentions)
11 protocols using alexa fluor 568 conjugated donkey anti mouse
1
Visualizing Biocytin-Labeled Neurons
2
Immunocytochemical Analysis of Neural Cell Markers
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Immunocytochemical staining was performed as previously described [33 (link)]. The primary antibodies included dendritic marker microtubule-associated protein 2 (MAP2, chicken, 1 : 4000, NB300-213, Novus), microtubulin marker β-tubulinIII (mouse, 1 : 1000, T8660, Sigma), apoptosis marker cleaved caspase-3 (cl-Casp3, rabbit, 1 : 400, 9664, Cell Signaling), and proliferation marker Ki-67 (rabbit, 1 : 800, AB9260, Millipore). The secondary antibodies included Alexa Fluor 488-conjugated donkey anti-rabbit (1 : 400), Alexa Fluor 568-conjugated donkey anti-mouse (1 : 400), and Alexa Fluor 647-conjugated goat anti-chicken (1 : 200, all from Thermo Fisher Scientific). Cell samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired with an Olympus IX51 microscope equipped with an Olympus DP30BW camera (Olympus Corporation). CellProfiler [34 (link)] and CellProfiler Analyst [35 (link)] were used for image analysis.
3
3D Visualization of Transplanted Interneurons
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To visualize the cIN grafts in 3D in mouse brains, we performed immunohistochemistry/tissue-clearing according to the iDISCO+ tissue clearing method 27 (link). The mouse was perfused with 0.1 M PBS followed by 4% PFA 4 months after transplantation. The brain was harvested and post-fixed in 4 % PFA solutions overnight. The mouse brain was dehydrated, bleached, rehydrated, and blocked as described 27 (link). The following antibodies were used for staining: mouse-anti-human cytoplasm (1:1,000, StemCells Inc. Newark, CA), and Alexa Fluor 568-conjugated donkey anti-mouse (1:1,000, Thermo Scientific). Solvent-cleared brain was imaged in its entirety with light-sheet microscopy (LaVision Ultramicroscope II Light Sheet, LaVision BioTec, Germany) at 2.5 X objective. Z-stack images taken from light-sheet microscopy were subjected to 3D rendering and spot analysis using Imaris software 9.2 (Bitplane).
4
Intracellular Integrin Trafficking Visualized
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Intracellular integrin trafficking was analyzed in HUVECs grown on 10 μg/ml EGFL7 and/or 4 μg/ml Fn‐coated coverslips. Subsequently, cells were stained for EEA1, Lamp‐1, and integrin α5β1 or αVβ3. In brief, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X‐100. After blocking, cells were stained with sheep anti‐EEA1 (AF8047, 1:100, R&D Systems, Minneapolis, MN, USA), rabbit anti‐Lamp‐1 (ab24170, 1:100, Abcam, Cambridge, MA, USA), mouse anti‐α5β1 (MAB1969, 1:100, Merck), or mouse anti‐αVβ3 (MAB1976, 1:100, Merck) primary antibodies. Following incubation with Alexa Fluor 488‐conjugated donkey anti‐rabbit (1:1,000, Thermo), Alexa Fluor 647‐conjugated donkey anti‐sheep (1:1,000, Thermo), or Alexa Fluor 568‐conjugated donkey anti‐mouse (1:1,000, Thermo), nuclei were counterstained by DAPI staining. The coverslips were mounted using Fluoromount‐G (SouthernBioTech, Birmingham, AL, USA), and images were taken using an SP8 confocal microscope (Leica, Mannheim, Germany). 3D reconstruction was performed using Imaris 8 (Bitplane, Zurich, Switzerland) and ImageJ software v1.41 (National Institute of Health, Bethesda, MD, USA).
5
Immunofluorescent Labeling of CYP1A2 and HIF-1α
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Cells were fixed by 4% paraformaldehyde (4% PFA/PBS) and permeabilized with 0.1% Triton X-100 at room temperature for 15 min. After blocking with 0.1% FBS, cells were blocked and immunolabeled with Anti-CYP1A2 (sc-53241) and Anti-HIF-1α (ab2185) at 4 °C overnight. On the following day, fluorescence-conjugated secondary antibodies, Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 568-conjugated donkey anti-mouse (both from Thermo Fisher), were stained at room temperature for 2 h. Nuclei were counterstained with DAPI (Invitrogen) for observation.
6
3D Visualization of Transplanted Interneurons
7
Immunohistochemical Analysis of Mouse Brain
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Shortly after the behavioral or electrophysiology experiments, mice were sacrificed and perfused transcardially with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. Brains were retrieved, fixed in 4% PFA for 24 hr before being placed in a 30% sucrose solution. Brains were sliced at 30 μm thickness and prepared for the appropriate incubations, as described previously50 (link). Primary antibody included rabbit anti-GFP (1:1000, Millipore 06-896), Mouse anti-HuC/D (1:200, Molecular Probes A21271), mouse anti-NeuN (1:100, Millipore MAB377), rabbit anti-TH (1:1000, Pel-Freez P40101-0), at room temperature for 24 hours or at 4°C for 48 h when using rabbit anti-PV (1:1000, Swant PV 27). Secondary antibodies included Alexa Fluor 488-conjugated goat anti-chicken (1:500, Life Technologies), Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Life Technologies), Alexa Fluor 568-conjugated donkey anti- rabbit (1:500, Life Technologies), Alexa Fluor 568-conjugated donkey anti-mouse (1:500, Life Technologies), Alexa Fluor 647-conjugated donkey anti-rabbit (1:500, Life Technologies), or Alexa Fluor 647-conjugated donkey anti-mouse (1:500, Life Technologies).
8
Immunocytochemistry Analysis of CMs
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Immunocytochemistry techniques were carried out as previously described.53 (link) CMs were allowed to adhere to laminin-coated coverslips (20µg/mL) at 37°C for 2 h and subsequently fixed in a 1:1 acetone/methanol solution for 30 minutes. After washing with phosphate-buffered saline, cells were blocked before addition of primary antibodies. After blocking has occurred, CMs were double stained with combinations of the following primary antibodies: anti-α-actinin, anti-vinculin (Upstate Biotechnology, Lake Placid, NY), rabbit anti-TRPA1 and/or mouse anti-TRPV1. Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 568-conjugated donkey anti-mouse (Life Technologies) were used as secondary antibodies. Negative controls included CMs incubated with a single primary antibody and both secondary antibodies, as well as CMs incubated with secondary antibodies alone. Images were acquired using an Olympus Fluoview 100 confocal laser scanning microscope with an X63 objective lens.All images were acquired utilizing a 60X objective with a gain setting of 500 V and laser excitation wavelengths of 488 nm (multi-line Ar, 8.0% full power, BA505-525 filter) and 543 nm (HeNe, 12.0% full power, BA560-660 filter).
9
Immunohistochemical Analysis of Mouse Brain
10
Antibody Characterization for Cell Biology
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The following antibodies were used: anti-EHD1 (109311, Abcam), affinity-purified rabbit polyclonal peptide antibody directed against the C-terminus of CORO2A (RELLTQREVQAKQLELEIKN; LifeTein for IP and immunoblotting), anti-TOM20 (sc-11415, Santa Cruz Biotechnology), anti-HA (T501, SAB), anti-FLAG (F1804, Sigma for immunofluorescence; ab205606, Abcam for immunoblotting), anti-GST-HRP (A01380, GenScript), anti-GAPDH-HRP (HRP-60004, Proteintech), anti-MHC-1 (purified W6/32, Leinco Technologies), anti-CD98 (315602, BioLegend), anti-CD59 (A4-233-C100, Exbio), anti-EEA1 (3288, Cell Signaling), anti-MICAL-L1 (H00085377-B01P, Novus), donkey anti-mouse-HRP (715-035-151, Jackson), mouse anti-rabbit IgG light chain-HRP (211-032-171, Jackson), Alexa Fluor 568–conjugated goat anti-rabbit (A11036, Molecular Probes), Alexa Fluor 568–conjugated donkey anti-mouse (21043, Molecular Probes), Alexa Fluor 488–conjugated goat anti-rabbit (A11034, Molecular Probes), and Alexa Fluor 488–conjugated donkey anti-mouse (A21202, Molecular Probes). The following reagents were used: CF-568–conjugated Phalloidin (44-T VWR, Biotium), cytochalasin D (Calbiochem), Alexa fluor 568–conjugated transferrin (Molecular Probes), Rhodamine-tagged EGF(Invitrogen), and Blasticidin S HCl (ThermoFisher Scientific).
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