Arterial myocytes were fixed, permeabilized, and exposed to anti-TRPV4 (H-79) and anti-AKAP150 (N-19) antibodies (Santa Cruz Biotechnology, Inc.). Alexa Fluor 488–conjugated donkey anti–goat (5 µg/ml) and an Alexa Fluor 568–conjugated donkey anti–rabbit (5 µg/ml; Molecular Probes) were used as secondary antibodies. Confocal images were acquired with a confocal microscope (Fluoview FV1000; Olympus) equipped with a UPlanS-Apochromat 60× (NA 1.2) water immersion objective lens. The resolution of our confocal microscope was ∼250 nm in the x-y axis and ∼800 nm in the z axis. Myocytes were imaged with a zoom of 3.5 (1,024 × 1,024 pixel images; pixel size = 0.1 µm). Alexa Fluor 488 and Alexa Fluor 568 were excited using 473 nm and 559 nm lasers, respectively. Fluorescence was quantified by measuring the intensity of pixels above a threshold defined as the mean nonspecific fluorescence intensity within cells (i.e., cell background, secondary antibody only) plus three times its SD. We calculated the Pearson’s coefficient of TRPV4 and AKAP150 images to quantify the degree of overlap between fluorophores within the confocal volume.
Alexa fluor 488 conjugated donkey anti goat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated donkey anti-goat is a fluorescent-labeled secondary antibody used in immunoassays and other biological applications. It binds to goat primary antibodies and emits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated donkey anti rat, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Antigen retrieval buffer, by Agilent Technologies (1 mentions)
Rabbit anti mpo, by Agilent Technologies (1 mentions)
Immunofluorescence microscope, by Leica (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Tc20 automatic cell counter, by Bio-Rad (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Cellinsight nxt, by Thermo Fisher Scientific (1 mentions)
Alexafluor 546 conjugated donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Triton x 100, by Nacalai Tesque (1 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (1 mentions)
Alexa fluor 555 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
6 protocols using alexa fluor 488 conjugated donkey anti goat
1
Colocalization of TRPV4 and AKAP150 in arterial myocytes
2
Immunofluorescent Liver Staining Protocol
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For immunofluorescent double staining of MPO/properdin and C3c/properdin, 4µm thick liver sections were rehydrated and antigen binding sites were retrieved using preheated (95°C) Antigen Retrieval Buffer (Dako) for 30′, followed by a cooling down period of 20′. Non-specific binding sites were blocked with 10% goat serum in PBS. Sections were incubated overnight with rabbit-anti-MPO (Dako; 1∶1000) and monoclonal anti-C3c (1∶200) or goat-anti-properdin (Nordic Immunology, 1∶250) and monoclonal anti-C3c (1∶200) at 4°C, rinsed with PBS, and subsequently incubated with Alexa Fluor 488-conjugated donkey anti-goat (Invitrogen Molecular Probes, Eugene, OR, 4µg/ml) for 1,5 h at room temperature, followed by an appropriate Cy3-conjugated IgG antibody (Invitrogen, 1∶500) for 1,5 h at room temperature. Nuclei were stained with 4′,6-diamino-2-phenyl-indol (DAPI), and sections were mounted with Fluorescent Mounting Medium (Dako) and observed with a Leica immunofluorescence microscope.
3
Single-Cell Immunophenotyping of Pancreatic Cells
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Cells were dissociated into single cells by gentle pipetting after treatment with 0.25% trypsin–EDTA (Invitrogen) for 15 min at 37 ℃ and fixed with a Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s protocol. Then, the cells were blocked with 2% donkey serum in permeabilization solution and incubated with the following primary antibodies diluted in blocking solution overnight at 4 °C: goat anti-PDX1 (R&D Systems, AF2419, 1:200), mouse anti-NKX6.1 (DSHB, F55A12, 1:200), rabbit anti-NKX6.1 (Cell Signaling, 54551, 1:500) and rat anti-CPEP (DSHB, GN-ID4, 1:200). After washing with wash buffer once, the cells were incubated with the following fluorescent secondary antibodies for an hour at room temperature: Alexa Fluor 488-conjugated donkey anti-goat (Thermo Fisher Scientific, A-11055, 1:500), Alexa Fluor 647-conjugated donkey anti-mouse (Thermo Fisher Scientific, A-31571, 1:500), Alexa Fluor 647-conjugated donkey anti-rabbit (Thermo Fisher Scientific, A-31573, 1:500) and Alexa Fluor 488-conjugated donkey anti-rat (Thermo Fisher Scientific, A-21208, 1:500). The stained cells were analyzed using a FACSAria II or LSRFortessa (BD Biosciences). The cell number was counted using a TC20™ automatic cell counter (Bio-Rad).
4
Immunofluorescence Staining of Pancreatic Cells
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Cells were washed with PBS (Nacalai Tesque) twice, fixed with 4% PFA (Nacalai Tesque)/PBS for 20 min at 4 °C and blocked in PBS with 5% donkey serum (Millipore) and 0.4% Triton X-100 (Nacalai Tesque) (blocking solution) for 30 min at room temperature. The following primary antibodies were diluted in blocking solution and incubated overnight at 4 °C: goat anti-PDX1 (R&D Systems, AF2419, 1:200), mouse anti-NKX6.1 (DSHB, F55A12, 1:200), rabbit anti-NKX6.1 (Cell Signaling, 54551, 1:500) and rat anti-CPEP (DSHB, GN-ID4, 1:200). After washing with PBS, the cells were incubated with the following fluorescent secondary antibodies for 1 h at room temperature: Alexa Fluor 488-conjugated donkey anti-goat (Thermo Fisher Scientific, A-11055, 1:500), Alexa Fluor 546-conjugated donkey anti-mouse (Thermo Fisher Scientific, A-10036, 1:500), Alexa Fluor 555-conjugated donkey anti-rabbit (Thermo Fisher Scientific, A-31572, 1:500) and Alexa Fluor 488-conjugated donkey anti-rat (Thermo Fisher Scientific, A-21208, 1:500). Immunofluorescence images were acquired using a CellInsight NXT (Thermo Fisher Scientific) or BZ-9000 (Keyence). For quantification, 9 images per well were obtained using CellInsight NXT.
5
Mouse Spinal Cord Immunohistochemistry
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The fixation, cryoprotection, and sectioning of mouse spinal cord were performed as previously described for rat spinal cords (Fig. 7, B to D ). After sectioning, adult C57Bl/6 mice spinal cord sections were dipped in 0.1 M PB and washed with 0.05 M TBS-Tx (1× for 10 min). Sections were further incubated in 2% NDS diluted in 0.05 M TBS-Tx (40 min at RT) and then in rabbit anti-nNOS antibody (1:6000; catalog no. ab229785, Abcam, Toronto, Canada) and goat anti-ChAT antibody (1:1000; catalog no. ab144P, Merck Millipore, Billerca, MA, USA) diluted in 0.05 M TBS-Tx for 48 hours at 4°C. Briefly, sections were washed in 0.05 M TBS-Tx (3x for 10 min) and further incubated in Alexa Fluor 594–conjugated donkey anti-rabbit (1:400; catalog no. A-21207, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488–conjugated donkey anti-goat (1:400; catalog no. A-11055, Thermo Fisher Scientific, Waltham, MA, USA) in 0.05 M TBS-Tx for 2 hours at RT. Sections were then washed in 0.05 M Tris-HCl (3× for 10 min), mounted onto slides coated with 0.1% gelatin (Sigma-Aldrich), and coverslipped using Fluoromount (H-1000, Vector Labs).
6
In Situ BDNF-Astrocyte Interaction
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For in situ detection of BDNF and astrocyte interaction, proximity ligation assay (PLA; Duolink, Millipore Sigma) was performed in fixed retinal flat mounts as described above following the manufacturer's instructions. Retinal flat mounts were permeabilized with 0.1% Triton X-100 in PBS. Retinal flat mounts were blocked in 5% normal donkey serum in PBS with 0.01% sodium azide and 0.3% Triton X-100 and primary incubations were in 5% normal donkey serum in PBS with 0.01% sodium azide. Retinal flat mounts were washed with PBS. Primary antibody incubations were performed for 24 hours at 4°C. Primary antibodies used for PLA detection were brain derived neurotrophic factor (BDNF; Novus Biologicals, 25928.11; 1:100 dilution) and glutamate transporter-1(GLT-1; Alomone, AGC-022; 1:100 dilution). Co-labeling for astrocytes was performed using the same anti-glial fibrillary acidic protein antibody described above (GFAP; Abcam, ab53554; 1:250 dilution). Secondary antibody incubation labeling GFAP positive cells was performed for 1 hour at room temperature using Alexa Fluor 488-conjugated Donkey anti-Goat (ThermoFisher, A-11055; 1:500 dilution) during the PLA probe incubation.
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