C2C12 cells in 24-well plates were fixed with 4% formaldehyde for 30 min and washed three times in PBS for 5 min each time. The fixed cells were incubated with 0.5% Triton X-100 in PBS for 10 min, 3% H2O2 for 10 min, the primary antibodies against myosin (MAB-0124, Maixin, Fuzhou, China) and CaMKIIδ (ab105502, Abcam, Cambrige, UK) overnight at 4 °C, and then, the secondary antibodies (Alexa Fluor 594, a11037, Invitrogen, Carlsbad, CA, USA) for 45 min before counterstained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI, D9542, Sigma, St. Louis, MO, USA) for 10 min. Images from at least three regions in each well were captured by a fluorescence microscope (DP72, Olympus, Japan). The fusion index was calculated as the ratio of the number of nuclei in myosin-positive myotubes with two or more nuclei to the total nuclei number.
4 6 diamidino 2 phenylindole dihydrochloride dapi d9542
Manufactured by Merck Group
Sourced in United States, France
4′ 6-Diamidino-2-phenylindole dihydrochloride (DAPI D9542) is a fluorescent dye commonly used in molecular biology and cell biology research. It is a nucleic acid stain that binds preferentially to adenine-thymine (A-T) base pairs in DNA, allowing visualization and detection of DNA in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Nnos 610308, by BD (2 mentions)
Ab105502, by Abcam (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β catenin, by BD (1 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (1 mentions)
Alexa fluor 568 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti vimentin, by Merck Group (1 mentions)
Mouse monoclonal anti acetylated α tubulin, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Triton x 100 x100, by Merck Group (1 mentions)
Bovine serum albumin a6003, by Merck Group (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
P6148, by Merck Group (1 mentions)
Stellaris 5, by Leica (1 mentions)
P0098, by Beyotime (1 mentions)
Bx51 fluorescent microscope, by Olympus (1 mentions)
Ab109290, by Abcam (1 mentions)
Ab128995, by Abcam (1 mentions)
C myc 10828 1 ap, by Proteintech (1 mentions)
8 protocols using 4 6 diamidino 2 phenylindole dihydrochloride dapi d9542
1
Immunofluorescence Staining of C2C12 Myocytes
2
Immunofluorescence Staining of Cytoskeletal Markers
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The following commercial antibodies and reagents were used: mouse monoclonal anti–α-tubulin (T9026); mouse monoclonal anti-vimentin (V6630); mouse monoclonal anti-acetylated α-tubulin (T6793); tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951); 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, D9542) (Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal anti–β-catenin (#610153; BD Biosciences, Franklin Lakes, NJ, USA); mouse monoclonal anti-YAP (SC-101199) and mouse monoclonal anti-phosphotyrosine (PY99, SC-7020) (Santa Cruz, Santa Cruz, CA, USA); rabbit polyclonal anti–α-tubulin (ab18251; Abcam, Cambridge, UK); mouse monoclonal anti-nestin (#33475; Cell Signaling, Danvers, MA, USA); rabbit polyclonal anti EB3 (AB6033; Merck-Millipore, Darmstadt, Germany); AlexaFluor 488-conjugated donkey anti-mouse (A21202); AlexaFluor 488-conjugated goat anti-rabbit (A11008); AlexaFluor568-conjugated goat anti-mouse (A11031); AlexaFluor568-conjugated donkey anti-rabbit (A10042) (Thermo Fisher Scientific, Waltham, MA, USA).
3
Immunofluorescence Staining Protocol
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We bought the following reagents from Sigma-Aldrich (St. Louis, MO, USA): 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) (D9542), bovine serum albumin (A6003), Fluoromount™ compound (F4680), paraformaldehyde (PFA) (P6148), phosphate-buffered saline (PBS) (P3813), Tween® 20 (P2287) and Triton X-100 (X100). Normal goat serum (NGS, 04-009-1A) was purchased from Biological Industries (Cromwell, CT, USA). BrdU (ab142567) was obtained from Abcam (Cambridge, UK). The list of antibodies used in this study can be found in Table 1 .
4
Immunohistochemical Analysis of Enteric Neurons
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Distal colon segments were fixed in 0.1M PBS containing 4% paraformaldehyde at room temperature for 3 h. Whole mounts of longitudinal muscle and myenteric plexus were obtained by microdissection and permeabilized with PBS containing 10% horse serum (HS) and 4% Triton X-100 for 2 h at room temperature. Tissues were then incubated with the following primary antibodies: rabbit anti-ChAT (1:1000, a gift from Professor M. Schemann, Hannover, Germany) (Schemann et al., 1993 (link)), mouse anti-neuronal NOS (nNOS; 610308, 1:500; BD Biosciences), rabbit anti-GR (D8H2, 3660S 1:500, Cell Signaling), human anti-Hu (Bodin et al., 2021 (link)) (gift from the CHU of Nantes; 1:5000) diluted in PBS containing 10% horse serum and 0.5% Triton X-100 for 24 or 48 h at room temperature. After washing, tissues were incubated for 2 h at room temperature with the appropriate secondary antibodies, respectively, anti-rabbit CY5 (1:500), anti-rabbit CY3 (1:500) and anti-human FP488 (1:200), and mounted Glycerol 60% (vol/vol) (Thermo Fisher Scientific). Nuclei were stained with 4′ 6-Diamidino-2-phenylindole dihydrochloride (DAPI D9542; 1:10000; Sigma Aldrich, Paris, France).
5
TUNEL Assay for Apoptosis in PTECs
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This assay was performed using an assay kit (12156792910, Sigma-Aldrich, Germany) [26 (link)]. PTECs with various interventions (5 × 104 cells/well) were seeded in 12-well plates and fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, Germany) at ambient temperature for 10 min, then permeabilized in 1% Triton X-100 (T8787, Sigma-Aldrich, Germany) for 5 min and covered with 50 mL of TdT enzyme reaction solution for 1-h incubation in a wet box. PTECs were further covered with 50 mL streptavidin-fluorescein labeling buffer at 37 °C for 30 min, and the nuclei were stained with 10 µL 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI, D9542, Sigma-Aldrich, Germany) for 5 min. A confocal laser scanning microscope (Stellaris 5, Leica, Wetzlar, Germany) was used to observe the cells at 200 × magnification.
6
Immunostaining of Adult Midguts
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Adult midguts were dissected in 0.85% NaCl, fixed at 4°C overnight in fixative solution (P0098, Beyotime), washed in PBST [PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100] 3 times, and then incubated in primary antibodies (at 4°C overnight) and secondary antibody (at 25°C for 4 h or 4°C overnight) prepared in PBST. All antibodies were listed in Supplementary Table 1 . Nuclei were counterstained with 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) (D9542, Sigma). All images were taken on the SP8 STED Confocal Microscope or Olympus BX51 fluorescent microscope. Images were processed and quantified using ImageJ software.
7
Optimizing Cell Proliferation and Protein Expression Analysis
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Doxycycline (HY-N0565B) and CFSE (HY-D0938) were purchased from Medchem Express (MCE). Bovine serum albumin (BSA, FC0077) and propidium iodide (PI, 219545810) were purchased from MP Biomedicals. RNase A (10406ES03) was purchased from Yeasen Biotechnology. 5-Bromo-2′-deoxyuridine (BrdU, B5002), Hoechst 33342 (B2261), Triton X-100 (T8787), Pyronin Y (213519), and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, D9542) were purchased from Sigma-Aldrich. Paclitaxel was purchased from Beijing SL Pharmaceutical Co., Ltd. (China). Cisplatin was purchased from Jiangsu Hansoh Pharmaceutical Co., Ltd. (China).
Antibodies against SOX1 (ab109290) and RPS3 (ab128995) were purchased from Abcam. Antibody against RPL7A (DF9137) was purchased from Affinity Biosciences. Antibodies against β-actin (60008-1-Ig), Ki-67 (27309-1-AP), and c-MYC (10828-1-AP) were purchased from Proteintech. Antibody against p27 Kip1 (sc-528) was purchased from Santa Cruz Biotechnology. Alexa Fluor 488 cross-adsorbed goat anti-rabbit IgG secondary antibody (A11008) was purchased from Invitrogen.
Antibodies against SOX1 (ab109290) and RPS3 (ab128995) were purchased from Abcam. Antibody against RPL7A (DF9137) was purchased from Affinity Biosciences. Antibodies against β-actin (60008-1-Ig), Ki-67 (27309-1-AP), and c-MYC (10828-1-AP) were purchased from Proteintech. Antibody against p27 Kip1 (sc-528) was purchased from Santa Cruz Biotechnology. Alexa Fluor 488 cross-adsorbed goat anti-rabbit IgG secondary antibody (A11008) was purchased from Invitrogen.
8
Immunohistochemistry of Myenteric Plexus
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Distal colon segments were xed in 0.1M PBS containing 4% paraformaldehyde at room temperature for 3h. Whole mounts of longitudinal muscle and myenteric plexus were obtained by microdissection and were permeabilized with PBS containing 10% horse serum (HS) and 4% Triton X-100 for 2h at room temperature. Tissues were then incubated with the following primary antibodies: rabbit anti-ChAT (1:1000, a gift from Professor M. Schemann, Hannover, Germany (32) , mouse anti-neuronal NOS (nNOS; 610308, 1:500; BD Biosciences), rabbit anti-GR (D8H2, 3660S 1:500, Cell Signaling), human anti-Hu (gift from the CHU of Nantes; 1:5000) diluted in PBS containing 10% horse serum and 0,5% Triton X-100 for 24 or 48h at room temperature. After washing, tissues were incubated for 2h at room temperature with the appropriate secondary antibodies, respectively, anti-rabbit CY5 (1:500), anti-rabbit CY3 (1:500) and antihuman FP488 (1:200), and mounted Glycerol 60% (vol/vol) (Thermo Fisher Scienti c). Nuclei were stained with 4′ 6-Diamidino-2-phenylindole dihydrochloride (DAPI D9542; 1:10000; Sigma Aldrich, Paris, France).
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