Immunocytochemistry was conducted as previously described [25 (link)]. For overnight incubation at 4 °C we employed primary antibodies against Ki67 (1:200, sc-23900, Santa Cruz Biotechnology, Dallas, TX, USA) or NRP1 (1:250, ab81321, Abcam, Cambridge, UK). Afterwards, cells were washed thrice with PBS and incubated for 1 h at room temperature with the secondary antibodies goat anti-rabbit conjugated with Alexa Fluor®647 (1:1000, ab150079, Abcam) or goat anti-mouse conjugated with Alexa Fluor®488 (1:1000, ab150113, Abcam). Then, the coverslips were washed three times with PBS, incubated with DAPI for nucleic acid staining (Sigma-Aldrich) for 5 min at room temperature, washed again three times with PBS and mounted on glass slides with the mounting medium Fluoromount-GTM (Thermo Fisher Scientific, Waltham, MA, USA). The immunocytochemistry (ICC) samples were visualized in a Zeiss LSM 800 confocal laser scanning microscope (Zeiss, Jena, Germany). The images were analyzed using Zeiss ZEN and Fiji/ImageJ software, employing the CTCF formula relative to cell number to represent results.
Dapi for nucleic acid staining
Manufactured by Merck Group
Sourced in United States
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye used for nucleic acid staining. It binds strongly to adenine-thymine (A-T) rich regions in DNA, emitting blue fluorescence when excited by ultraviolet (UV) or violet light. DAPI is commonly used in various biological and microscopy applications to visualize and identify cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Ab150079, by Abcam (1 mentions)
Ab81321, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (1 mentions)
Sc 23900, by Santa Cruz Biotechnology (1 mentions)
Fluoromount gtm, by Thermo Fisher Scientific (1 mentions)
Kt5720, by Bio-Techne (1 mentions)
Forskolin, by Merck Group (1 mentions)
Anti creb 48h2, by Cell Signaling Technology (1 mentions)
N6 2 phenylisopropyl adenosine r pia, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit antibody, by Merck Group (1 mentions)
Poly 1 c hmw tlr 3 ligand, by InvivoGen (1 mentions)
Dibutyryl camp db camp, by Merck Group (1 mentions)
Cpg odn 2395, by InvivoGen (1 mentions)
Pam2csk4, by InvivoGen (1 mentions)
Anti gf of rsv, by US Biological (1 mentions)
Pe anti mcd47 miap301, by BioLegend (1 mentions)
Trustain fcx anti mouse cd16 32, by BioLegend (1 mentions)
Anti sca 1 d7, by BioLegend (1 mentions)
Cd326 g8, by BioLegend (1 mentions)
3 protocols using dapi for nucleic acid staining
1
Immunocytochemical Analysis of Ki67 and NRP1
2
Toll-Like Receptor Signaling Assays
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LPS (TLR4 ligand) from Escherichia coli serotype 055:B5, (−)-N6-(2-Phenylisopropyl) adenosine (R-PIA), dibutyryl cAMP (dbcAMP) and forskolin were obtained from Sigma. Pam2CSK4 (TLR2/TLR6 ligand), poly(I:C)-HMW (TLR 3 ligand), imiquimod (TLR7 ligand) and CpG ODN 2395 (TLR9 ligand) were purchased from InvivoGen. Resiquimod (TLR7/8 ligand) was kindly provided by Dr. Marianela Candolfi (INBIOMED-UBA-CONICET). Imiquimod was dissolved in water, according to the manufacturer's instructions. The rabbit monoclonal anti-Phospho-CREB (Ser133) (87G3) and anti-CREB (48H2) were purchased from Cell Signaling. KT5720 was obtained from Tocris Bioscience. The mouse monoclonal antibody anti-gF of RSV was obtained from US Biological Life Sciences. Secondary goat anti-mouse FluoroLinkTM CyTM3 antibodies were purchased from GE Healthcare. The peroxidase-conjugated goat anti-rabbit antibodies and Dapi for nucleic acid staining were obtained from Sigma.
3
Multimodal Isolation of Murine Organ Cells
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The lung, liver, spleen, kidney, and heart were extracted after mice were perfused with 1X PBS via the right atrium. For the heart, we used collagenase IV (10 mg/mL) as a digestive enzyme. For the spleen, no digestive enzymes were needed. The other tissues used collagenase type I, collagenase XI, and hyaluronidase. For the heart, we used collagenase IV. The cell suspensions were filtered through a 70-μm sterile nylon mesh cell strainer, washed with 1X PBS, and transferred to Eppendorf tubes. Fc receptors were blocked using TruStain fcX™ anti-mouse CD16/32 (BioLegend). The following antibodies were used: clone 6D5, 17A2, N418, M1/70, FA11, 30-F11, and 390. We also used anti-TER-119 (TER-119, BioLegend), anti-Annexin V (BioLegend), DAPI for nucleic acid staining (Sigma-Aldrich), PE anti-mCD47 (miap301, BioLegend), CD326 (G8.8, BioLegend), anti-Sca-1 (D7, BioLegend), anti-CD24 (M1/69, BD Biosciences), anti-CD271 (ME20.4, Invitrogen), and Monorab™ Rabbit Anti-Camelid VHH antibody, mAb (96A3F5, GenScript). Each stain was added at a 1:200 dilution to the cell suspension. Flow gating is shown (SI Appendix, Figs. S18 and S19 ). When Ai14 mice were used to quantify delivery, we also used PBS-treated Ai14 mice to control for flow gating.
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