DNA damage was analyzed using the single-cell gel electrophoresis (SCGE) alkaline comet assay, based on the protocol described by Sing and Stephens [21 (link)]. hPLFs exposed to 0.3 and 3 μM were detached after 24 h MeHg exposure, and the formed pellet was resuspended into 300 μL of new cell culture medium. An aliquot (20 μL) was homogenated with 120 μL of low-melting agarose and added to the slides pretreated with agarose layer. After drying, slides were incubated in lyse solution (in M: 2.5 NaCl, 0.1 EDTA, 0.01 Tris, 1% Triton X-100) and maintained overnight at 4°C. Following that, slides were placed into the electrophoresis solution (in mM: 300 NaOH, 1 EDTA; pH 13) for 20 minutes for the unwinding of the DNA. Electrophoresis was performed for 20 minutes at 30 V (1 V/cm) and 300 mA. The last steps were to neutralize the slides using 0.4 M Tris buffer (pH 7.5), stain them with DAPI (Enzo Life Sciences, NY, USA), and analyze them using a fluorescence microscopy (Zeiss Imager Z2, connected to the software Axiovison 4.8, Zeiss, Alemanha). One hundred cells per sample were automatically analyzed through Komet Software®. DNA damage was expressed as the length of the comet tail in percentage.
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Enzo Life Sciences
Sourced in United States, Italy
DAPI is a fluorescent dye used to stain DNA. It binds strongly to adenine-thymine (A-T) rich regions in DNA, allowing visualization of nuclei and chromosomes. DAPI is commonly used in fluorescence microscopy and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
E800 upright microscope, by Nikon (2 mentions)
Rat tail collagen 1, by Santa Cruz Biotechnology (2 mentions)
Rhodamine or fluorescein conjugated phalloidin, by Biotium (2 mentions)
Guinea pig anti vglut1, by Synaptic Systems (2 mentions)
Fluoromount, by Merck Group (2 mentions)
Hc pl apo 40 1.30 oil cs2, by Leica (2 mentions)
Tcs sp8 system, by Leica (2 mentions)
Imager z2, by Zeiss (1 mentions)
Axiovison 4, by Zeiss (1 mentions)
Pcreb, by Merck Group (1 mentions)
Anti chat, by Merck Group (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Alexa fluor 488 or 647, by Abcam (1 mentions)
Anti cd147, by Thermo Fisher Scientific (1 mentions)
Anti nrp1, by Thermo Fisher Scientific (1 mentions)
Anti beta 3 tubulin, by Abcam (1 mentions)
Anti smi 312, by Fortrea (1 mentions)
Calponin 1, by Abcam (1 mentions)
Cardiac troponin t ctnt, by Santa Cruz Biotechnology (1 mentions)
Pe conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
14 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Comet Assay for DNA Damage Analysis
2
Encapsulated Cell Viability in Collagen Hydrogels
3
Encapsulated Cell Viability in Collagen Hydrogels
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Alginate microgels encapsulating cells were themselves encapsulated in a bulk collagen hydrogel. Following manufacturer's instructions, rat tail collagen I (Santa Cruz Biotechnology, Santa Cruz, CA) was first mixed with Dulbecco's phosphate buffered saline and sodium hydroxide to achieve a neutral pH, and then mixed with a suspension containing cells encapsulated in alginate microgels to obtain a final collagen concentration of 1.85 mg/mL. The suspension was added to wells in a 48-well plate and allowed to cross-link at 37 C for 30 min. Collagen gels were fixed after 1 or 3 days of culture. Cells were stained with rhodamine- or fluorescein-conjugated phalloidin (Biotium, Hayward, CA) and DAPI (Enzo Life Sciences, Plymouth Meeting, PA), and imaged with a Nikon E800 upright microscope. Only microgels that showed a morphology consistent with having previously contained cells (i.e. hollowed out morphology) were considered to have led to cell-egress.
4
Immunofluorescence Staining of Brain Slices
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Brains were removed immediately after perfusion and postfixed in 4% paraformaldehyde overnight at 4°C. The day after, brains were cryoprotected in 30% sucrose in PBS. Coronal sections of 40 μm were prepared for immunofluorescence. Before incubation with primary antibodies, slices were washed in cold methanol (10 minutes, methanol 100%) and then further washed in PBS 1X to improve the detection of PSD 95 signal (as recommended in http://www.cellsignal.com/contents/resources-protocols/immunofluorescence-protocol-with-methanol-permeabilization-%28if-methanol-perm%29/if-methanol ). Brain slices were then incubated with primary antibodies in PBS 1X, 3% triton, at 4°C. Primary antibodies used were to PSD 95 (Anti-Rabbit, 1 : 100, o/we; Abcam), vesicular glutamate transporter 1 (vGlut1 Anti-Guinea Pig, 1 : 800, o/we; Synaptic System), phosphorylated CREB (pCREB, Anti-Rabbit, 1 : 1000, o/n; Millipore), and CREB (CREB, Anti-Rabbit, 1 : 600, o/n; Cell Signaling). Slices were then washed three times with PBS 1X and incubated with appropriate fluorescently labelled secondary antibodies (room temperature, dark, 2 hours). Slices were further washed three times in PBS 1X and then were counterstained with DAPI (1 : 1000, 10 minutes; Enzo life Science) after the final PBS wash. At last, sections were mounted with Fluoromount (Sigma Aldrich) and cover-slipped.
5
Immortalized Human HSC Characterization
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The immortalized human HSC were inoculated onto overlaps (Fisher, USA) in six-well plates. After 24 h of culture, the immortalized HSC attached onto the overlaps were washed twice with PBS. The overlaps were transferred and incubated in cold acetone for 10 min of fixing. Next, the overlaps were washed twice with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 15 min, and then washed twice with PBS. Then, the overlaps were incubated in blocking solution at 37 °C for 30 min and PBS containing 3% BAS with primary antibody for 120 min. After washing with PBS, the overlaps were incubated with a secondary antibody in PBS containing 3% BAS for 60 min. The overlaps were finally washed twice with PBS, and DAPI (ENZO, USA) was used for cell nucleus staining. The overlaps were visualized by fluorescence microscopy (Olympus, Japan). The primary antibodies included anti-human vimentin antibody (R&D Systems, USA), anti-human α-smooth muscle actin (α-SMA) antibody (R&D Systems, USA), platelet-derived growth factor receptor-beta (PDGFR-β) (Santa Cruz, USA) and mouse monoclonal anti-SV40LT antibody (Santa Cruz, USA). The secondary antibodies included FITC fluorescent goat anti-mouse antibody and donkey anti-rabbit antibody (Abcam, USA).
6
Evaluating SARS-CoV-2 Receptor Expression in iPSC-Derived Motor Neurons
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Induced Pluripotent Stem Cells-MNs were seeded on coverslips in a 24-well plate, cultured until differentiation, and infected as specified above. At 48 hpi, cells were fixed in PBS containing 4% PFA at RT for 10 min, followed by permeabilization with 0,1% TritonX-100 in PBS for 10 min. Cells were treated with 1% BSA in PBS for blocking at RT for 1 h, and incubated at 4°C overnight with specific primary antibodies. The following primary antibodies were used: anti-beta III Tubulin (1:500, Abcam, Cambridge, UK), anti-SMI-312 (1:1000, Covance, Princetown, NJ, USA) and anti-ChAT (1:200, Chemicon) to assess iPSC-MN differentiation; anti-ACE2 (1:200, Prodotti Gianni), anti-CD147 (1:100, Thermo Fisher Scientific) anti-NRP1 (1:100, Thermo Fisher Scientific) and anti-N Nucleocapsid SARS-CoV-2 (1:1000, BEI Resources) to assess SARS-CoV-2 receptors and infection. Coverslips were then stained with secondary antibodies (Alexa Fluor 488 or 647, 1:500, Abcam) for 45 min at RT and mounted using a medium containing DAPI (Enzo Life Sciences, Milan, Italy). Confocal images were acquired on a TCS SP8 System equipped with a DMi8 inverted microscope and a HC PL APO 40 × /1.30 Oil CS2 (Leica Microsystems, Wetzlar, Germany) at a resolution of 1024 × 1024 pixels (single stack).
7
Cardiac Lineage Marker Analysis in Stem Cells
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Cells from P7, P28, and P53 were analyzed with IF staining for the surface markers Sca-1 and CD31. Induced and control group cells were labeled with IF for cardiac cell lineage-specific markers. Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature (RT), permeabilized if noted with 0.3% Triton X-100 (Sigma) for 20 min at RT, washed with PBS, blocked with 10% donkey or rabbit serum (Multisciences Biotech, China) in PBS for 30 min at 37°C, and then incubated for 2 hours at RT with primary antibodies against Sca-1 (Millipore), CD31 (eBioscience), cardiac troponin T (cTNT; Santa Cruz), cardiac-myosin heavy chain (cMHC; Abcam), smooth muscle actin (SMA; Epitomics), smooth muscle-myosin heavy chain (sMHC; Abcam), or calponin-1 (Abcam). After rinsing with PBS, cells were subsequently incubated with Alexa Fluor 488-conjugated donkey or rabbit-originated secondary antibodies (Life Technologies, Invitrogen) or PE conjugated streptavidin (eBioscience) for 30 min at RT. Nuclei were counter-stained with DAPI (1:1000; Enzo) in PBS for 1 min at RT. Immunostaining was observed and photoed by an inverted fluorescence microscope (Leica DMI3000B, Germany) and analyzed by Image-Pro Plus 7.0 software (Media Cybernetics, America).
8
Thioflavin-S staining of amyloid in CaLu-3 cells
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After the incubation with the secondary antibodies, and following 3 × 5 min washes in PBS, CaLu-3 cells were incubated for 15 min at RT in the dark with a solution of 0.05% w/v Thioflavin-S (T1892, Merck-Sigma, Milan, Italy), which was freshly prepared in 50% ethanol/water and 0.22 μm filtered. Cells were washed twice with 50% ethanol for 10 min each, and then washed once with 80% ethanol for 20 min. Eventually, cells were washed in PBS, briefly rinsed with water, and coverslips were mounted on Superfrost glass slides using a mounting medium with DAPI (Enzo Life Sciences, Milan, Italy). Confocal images were acquired on a TCS SP8 System equipped with a DMi8 inverted microscope and a HC PL APO 40 × /1.30 Oil CS2 (Leica Microsystems, Wetzlar, Germany) at a resolution of 1024 × 1024 pixels.
9
Quantifying TFEB Nuclear Localization
10
Characterization of Spinal Cord Injury in Mice
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At day 21 post injury, the mice were anesthetized and perfusion-fixed with 4% paraformaldehyde (PFA) in PBS. The spinal cord tissues, including injured regions, were immersed in 30% sucrose solution. Spinal cords were sagittally sectioned at a thickness of 14 µm using a CM1860UV cryostat (Leica, Heidelberg, Germany). The sections were immunostained with a chicken anti-NF-H IgY polyclonal antibody (1:1,000; Cat. No. AB5539, Chemicon, Temecula, CA, USA). Alexa Fluor 488-conjugated goat anti-chicken IgY (1:400; Cat. No. A-11039, Life Technologies) was used as a secondary antibody. To decide the lesion area, nuclei were counterstained with 1 µg/ml DAPI (Enzo Life Science). Fluorescence images were obtained using an inverted microscope system (BZ-X710; Keyence, Osaka, Japan) with a 20× NA 0.75 objective lens (CFI PlanApo-λ; Nikon Instech, Tokyo, Japan). The injured regions were defined by the areas that accumulated DAPI-positive nuclei. The area of the injured region and the length of NF-H-positive axons were measured using MetaMorph version 7.8 (Molecular Devices).
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