Image acquisition of LUHMES cells live-stained with 1 µg/mL Hoechst H-33342 (Sigma Aldrich, Steinheim, Germany) and 1 µM calcein-AM (Biomol GmbH, Hamburg, Germany) was conducted by an automated high-content imager (Cellomics VTI, Waltham, MA, USA), as described previously [1 (link),2 (link),30 (link)].
Hoechst h33342
Manufactured by Merck Group
Sourced in Germany, United Kingdom
Hoechst H33342 is a fluorescent dye used in various laboratory applications. It binds to DNA and emits blue fluorescence when excited by ultraviolet or blue light. The dye can be used for staining and visualization of cell nuclei in a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab170183, by Abcam (2 mentions)
Fitc labelled secondary antibody, by Zhongshan Biotechnology (2 mentions)
Dmi4000b, by Leica (2 mentions)
Cellomics arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab24590, by Abcam (1 mentions)
A0082, by Agilent Technologies (1 mentions)
Sc 6251, by Santa Cruz Biotechnology (1 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Millicell ez slide, by Merck Group (1 mentions)
Ab31526, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Abcam (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
18 protocols using hoechst h33342
1
Imaging LUHMES cells with Hoechst and Calcein
2
Immunofluorescence Analysis of Extracellular Matrix
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Cells were fixed with 4% of paraformaldehyde (PFA). Immunolocalization of F-actin was performed with Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific Cat# A12381, RRID:AB_2315633 Waltham, MA, USA). Cell nuclei were stained with Hoechst H33342 (B2261 Sigma) and images were acquired using the Cellomics ArrayScan VTI HCS Reader (Thermo Scientific). For extracellular matrix analysis, endogenous peroxidase activity was blocked and overnight incubation at 4 °C with antibodies against type III Collagen (Cell Sciences Cat# PS062, RRID:AB_2245094), type I Collagen (Cell Sciences Cat# PS060, RRID:AB_420256), type IV Collagen (Santa Cruz Biotechnology Cat# sc-59814, RRID:AB_1121796), Fibronectin (Santa Cruz Biotechnology Cat# sc-8422, RRID:AB_627598) or Laminin (Thermo Fisher Scientific Cat# PA1-16730, RRID:AB_2133633) was followed by incubation with a horseradish peroxidase-coupled secondary antibody (peroxidase-conjugated AffiniPure F(ab′)2 fragment anti-IgG (Jackson ImmunoResearch Labs Cat# 111-036-006, RRID:AB_2313586). Peroxidase staining was obtained with 3,3-diaminobenzidin/H2O2 solution. Cells were counterstained with Masson’s Hemalun and images were acquired using a Zeiss microscope.
3
Immunofluorescence Staining of Vascular Markers
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The sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, and then incubated in 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 30 min at room temperature. The sections were then incubated with the primary antibodies in 2% BSA in a humidified chamber overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-human Von Willebrand factor antibody 1:100 (A0082, Dako, Milan, Italy); mouse monoclonal anti-CD31 antibody 10 μg/mL (ab24590, Abcam, Cambridge, UK); mouse monoclonal anti-FLK-1 antibody 1:100 (sc-6251, Santa Cruz Biotechnology Inc., Paso Robles, CA, USA); and mouse monoclonal anti-OB antibody 1:100 (sc-28344, Santa Cruz Biotechnology, Inc.). Immunofluorescence staining was performed for 1 h at room temperature with the secondary antibody DyLight 488-labeled anti-mouse IgG (KPL, Gaithersburg, MD, USA) or DyLight 549-labeled anti-rabbit IgG (H + L) (KPL) diluted to 1:1000 in 2% BSA. Nuclear staining was performed with 2 µg/mL Hoechst H33342 (Sigma-Aldrich) for 2 min. The sections were coverslipped with a drop of mounting medium.
4
Immunofluorescence Staining of Actin Cytoskeleton
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Cells were fixed in 4% paraformaldehyde in PBS for 10 min and then incubated in 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 30 min at room temperature. They were then incubated with primary antibodies in 2% BSA solution in a humidified chamber for 12 h at 4 °C. The rabbit polyclonal antihuman phalloidine antibody (Millipore Corporation, MA, USA) was the primary antibody. Immunofluorescence staining was performed using the secondary antibody DyLight 549-labeled anti-rabbit IgG (H + L) (KPL, Gaithersburg, MD, USA) diluted 1/1000 in 2% BSA for 1 h at room temperature. Nuclear staining was performed with 2 μg/mL Hoechst H33342 (Sigma-Aldrich) solution for 2 min.
5
Immunofluorescence Staining of Endothelial Cells
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The samples were incubated in 2% bovine serum albumin (BSA, Sigma Aldrich, St. Louis, MI, USA) solution in PBS for 30 min at room temperature. The sections were then incubated with the primary antibodies in 2% BSA solution in a humidified chamber overnight at 4 °C. The following primary antibodies were used: mouse anti-CD31 antibody; rabbit monoclonal anti-vinculin antibody; mouse polyclonal anti-vonWillebrand factor antibody. Immunofluorescence staining was performed with secondary antibodies anti-mouse IgG DyLight 488 labeled and anti-rabbit IgG (H + L) rhodamine (TRITC)-conjugated in 2% BSA for 1 h at room temperature. Nuclear staining was performed with 2 μg/mL Hoechst H33342 (Sigma-Aldrich, St. Louis, MI, USA) solution for 2 min [31 (link)].
6
Immunofluorescence Staining of Actin and VEGF
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Cells were fixed in 4% paraformaldehyde in PBS for 10 min, then incubated in 2% bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 30 min at room temperature. Cells were then incubated with primary antibodies in 2% BSA solution in a humidified chamber for 12 h at 4 °C. The following primary antibodies were used: rabbit polyclonal antihuman phalloidine antibody (Millipore Corporation, Boston, MA, USA) and mouse polyclonal anti-human VEGF antibody (Sigma-Aldrich, Boston, MA, USA). Immunofluorescence staining was performed using the secondary antibodies DyLight 549-labeled anti-rabbit IgG (H + L) (KPL, Gaithersburg, MD, USA diluted 1/1000 in 2% BSA for 1 h at room temperature. Nuclear staining was performed with 2 μg/mL Hoechst H33342 (Sigma-Aldrich) solution for 2 min.
7
Immunofluorescence Staining of Human and Canine Cells
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For immunofluorescence staining, 2 × 104 human and canine cells at p1, p3, and p5 were seeded on glass coverslips put into 24-well plates and cultured in BM. Twenty-four h after seeding, cells were fixed in 4% paraformaldehyde solution in PBS for 10 min, then permeabilized for 10 min in 0.5% Triton X-100 (Sigma-Aldrich) prepared in PBS. After three washing with PBS, the cells were incubated in 2% bovine serum albumin (BSA; Sigma-Aldrich) solution in PBS for 1 h at RT. The cells were then stained with 5 µg/mL phalloidin tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) for 40 min at RT. Nuclear staining was performed with 2 μg/mL Hoechst H33342 (Sigma-Aldrich) solution for 15 min. The cells were observed with the LeicaDM5000 B microscope (Imaging Facility, Department of Biology, University of Padova, Padova, Italy).
8
Immunostaining of Focal Adhesions
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Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (Invitrogen) for 15 min. and permeabilized with 0.1% Trition X-100 (Thermo Scientific) for 10 min. at room temperature. 5% FBS (Gibco Invitrogen), and 5% Bovine Serum Albumin (Sigma Aldrich), in phosphate buffered saline was used to block non-specific binding for 1 hour. Primary antibody, anti-vinculin (Sigma Aldrich, cat. no. V9131) at 1∶500, and secondary antibody Alexa-Fluor goat-anti-mouse 488 (Molecular Probes-Invitrogen, cat. no. A1101) at 1∶800 was used to label the focal adhesions. Hoechst H33342 (Sigma Aldrich) and Phalloidin-tetramethylrhodamine (Fluka) were used for staining the nuclei and actin-filaments, respectively.
9
Validating Stem Cell Protein Expression
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From the nucleoprotein and membrane protein lists, we validated the expression of several proteins involved in stem cell function. SSCs were cultured on MEF feeders in a Millicell EZ slide (Millipore Corporation). SALL1, EZH2, RCOR2 and PLD6 were chosen for further exploration based on previous reports. The following commercial antibodies were used: rabbit anti-SALL1 (ab31526; Abcam, Cambridge, MA, USA), mouse anti-EZH2 (612666; BD Bioscience, San Jose, CA, USA), mouse anti-RCOR2 (612146; BD Bioscience) and rabbit anti-PLD6 (ab170183; Abcam). For the controls, the primary antibodies were replaced with normal rabbit or mouse IgG. After being washed with PBS, the samples were incubated with FITC-labelled secondary antibody (Beijing ZhongShan Biotechnology). The nucleus was stained with 5 μg/ml Hoechst H33342 (Sigma-Aldrich). All samples were observed under a ZEISS LSM 710 (Carl Zeiss).
10
Immunofluorescent Analysis of Pluripotency Markers
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The three primary antibodies used against SSC markers were based on previous reports: goat anti-OCT3/4 (sc8629; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-PLZF (OP128L; Millipore Corporation) and goat anti-GFRA1 (GT15004; Neuromics, Edina, MN, USA). Mouse anti-SSEA1 (sc-101462; Santa Cruz Biotechnology) was used as a negative marker because SSEA-1 is an early germline development pluripotent marker primarily expressed in primordial germ cells 24 (link). For the controls, the primary antibodies were replaced with normal goat or mouse IgG. After they were washed with PBS, the cells were incubated with FITC-labelled secondary antibody (Beijing ZhongShan Biotechnology, Beijing, China). The nucleus was stained with 5 μg/ml Hoechst H33342 (Sigma-Aldrich), and the samples were analysed under a ZEISS LSM 710 (Carl Zeiss, Oberkochen, Germany).
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