Chloral hydrate was obtained from Abbott Laboratories (North Chicago, IL, USA). FITC-dextran (3–5 kDa), dimethyl sulfoxide (DMSO), Triton X-100 and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alizarin carmine was obtained from Chroma (Stuttgart, Germany). Texas Red-X phalloidin and the rabbit polyclonal antibody against ZO-1 were purchased from Zymed-Invitrogen (Carlsbad, CA, USA). Mouse monoclonal antibodies against Na,K- ATPase were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and the rabbit monoclonal antibody against Ki67 was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG were purchased from Invitrogen-Gibco (Carlsbad, CA, USA). Hoechst 33342 and BSA were obtained from Vector Laboratories (Burlingame, CA, USA).
Hoechst 33342
Manufactured by Vector Laboratories
Sourced in United States
Hoechst 33342 is a fluorescent dye that binds to DNA. It is commonly used in cell biology applications to visualize and stain nucleic acid.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Triton x 100, by Merck Group (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Chloral hydrate, by Abbott (1 mentions)
Bovine serum albumin (bsa), by Vector Laboratories (1 mentions)
Tcs sp5, by Leica (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab23981, by Abcam (1 mentions)
Normal goat serum (ngs), by Beyotime (1 mentions)
X71 u rfl t fluorescence microscope, by Olympus (1 mentions)
Concanavalin a (cona), by Vector Laboratories (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Secureseal adhesive, by Electron Microscopy Sciences (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Vector Laboratories (1 mentions)
Lsm 710, by Zeiss (1 mentions)
9 protocols using hoechst 33342
1
Immunofluorescence Staining of Epithelial Cells
2
Immunostaining of Retinal Ganglion Cells
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On day 7 after primary culture, RGCs plated on coverslips were fixed in 4% paraformaldehyde for 20 minutes and permeabilized with PBS supplemented with 0.1% Triton X-100 at room temperature. Subsequently, nonspecific blinding was blocked with 4% goat serum for 25 minutes at room temperature. Cells were then incubated with rat anti-mouse Thy1 monoclonal antibody (1:300; Abcam Inc., Cambridge, MA, USA) at 4°C overnight to identify neurons. Next, cells were washed with PBS and incubated with a red fluorescent protein-labeled goat anti-rabbit secondary antibody at room temperature for 2 hours (1:500; BD Biosciences, San Jose, CA, USA). Finally, cells were washed three times in PBS, with Hoechst 33342 (Vector Laboratories, Burlingame, CA, USA) used for cell nuclei staining. Images were visualized and cell numbers of positive staining and Hoechst staining counted using a confocal laser microscope (TCS, SP5; Leica, Mannheim, Germany) at 200× magnification.
3
Immunofluorescence analysis of osteogenesis markers
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Cells attached to HAS were suspended by culturing in a 0.5 mM EDTA/PBS solution for 15 min at 37°C and reseeded in 6-well plates (5×104 cells/well) for 24 h attachment at 37°C. The cells were fixed with 4% paraformaldehyde in 1X PBS containing 0.1% Triton X-100 for 30 min at room temperature, and non-specific binding was blocked with 10% normal goat serum (Beyotime Institute of Biotechnology) in 1X PBS for 1 h at room temperature. The cells were incubated at room temperature for 1 h with the following primary antibodies targeted against osteogenesis-associated proteins: Runt-related transcription factor 2 (RUNX2; cat. no. ab23981; 1:1,000; Abcam) and osteocalcin (OCN; cat. no. ab93876; 1:1,000; Abcam), osteopontin (OPN; cat. no. ab192143; 1:1,000; Abcam). Subsequently, the cells were incubated with an AlexaFluor 488-conjugated secondary antibody (cat. no. ab6702; 1:5,000; Abcam) for 1 h at room temperature. For TOM20 staining, an Alexa Fluor647-conjugated anti-TOM20 antibody was used (cat. no. ab209606; 1:200; Abcam) at room temperature for 2 h. The cells were mounted in Vectashield® and stained with Hoechst 33342 (Vector Laboratories, Inc.; Maravai LifeSciences) at room temperature for 10 min. Cells were imaged using an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation; magnification, ×100).
4
Biofilm Staining and Visualization
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Biofilms cultured on Aclar film were rinsed 3× using KPBS and stained for 15 min at room temperature with either 5 µg/ml Hoechst 33,342 and 20 ng/µl fluorescein-labeled Concanavalin A (Vector Laboratories, Burlingame, CA), or 2.5 µM Syto9 and 16 µM PI. Other lectins tested include fluorescein-labeled Soybean (SBA), wheat germ (weak interaction with parent and ΔepaOX [WGA]), Dolichos biflorus (DBA), Ulex europaeus (UEA 1), Ricinus communis (RCA120), Peanut (PNA), and Banana lectin (weak interaction with parent [BanLec]). All stains were purchased from Molecular Probes/ThermoFisher Scientific unless stated otherwise. Immunolabeled samples were either directly visualized by wet-mount or fixed in 2% paraformaldehyde in KPBS overnight at room temperature and mounted. Prolong diamond antifade mountant (ThermoFisher Scientific) was added to fixed Aclar on slides with a cover glass spacer to avoid damaging the biofilm structure (SecureSeal Adhesive, Electron Microscopy Sciences). Images were captured with a Zeiss AX10 using Zen 2.1 software as a wide-field snapshot or z-stack with a 20 × 0.8 numerical aperture (NA) or 100 × 1.3 NA objective. The images presented are maximum intensity projections obtained using the Fiji ImageJ package.48 (link)
5
Quantification of Mitochondrial BAX Translocation
6
Quantifying Cellular RNA Dynamics
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To analyze the level of RNA inside cells, confocal microscopy was performed using a Click-iT® RNA Alexa Fluor® 488 Imaging Kit (Molecular Probes). A549 cells grown on coverslips (5 × 104 cells/well in a 24-well plate) were incubated with 1 mM 5-ethynyl uridine (EU) for 20 h at 37 °C, allowing the incorporation of EU into newly synthesized RNAs. Cells were fixed and permeabilized as described above and then treated with 500 μl of 10 μM RNase A or 3D8 scFv antibody prepared in PBS containing 1 mM MgCl2 (pH 7.2) for 2 h at 37 °C. Cells were incubated with Alexa 488-azide solution, which ligates EU, for 30 min at RT in the dark, and washed three times with RNase-free PBS. Nuclei were stained with Hoechst 33342 (Vector Laboratories) for 30 min at RT. Images of intracellular green fluorescence were acquired by confocal microscopy (Carl Zeiss LSM 710).
7
Sphere Culture Immunofluorescence Assay
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Sphere culture-enriched lung cancer cells (1×106 cells) were plated onto slides and incubated overnight. The slides were incubated with paraformaldehyde fixation (4%) for 30 minutes at 4°C. Then, the slides were blocked with FBS for 1 hour. The primary antibodies were applied at a 1:100 dilution for 1 hour at room temperature. Phosphate-buffered saline was used as the negative control to replace the primary antibody and to exclude nonspecific binding. The slides were treated with corresponding immunofluorescence-labeled secondary antibodies at 37°C1 for 15 minutes. Cell nucleus was stained with Hoechst 33342 and mounting medium was added before confocal microscopy examination. All the primary antibodies, including Oct4, NANOG, SOX2, KLF5, β-catenin, and MYC, were purchased from Abcam (Cambridge, UK). The immunofluorescence-labeled secondary antibodies and Hoechst 33342 were purchased from Vector Laboratories, Inc (Burlingame, CA, USA).
8
Immunofluorescence Staining of Fixed FDB Fibers
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Fixed FDB fibers were blocked for 2 h at room temperature in PBS containing 5% bovine serum albumin, 1% normal goat serum, and 0.04% saponin, except for staining with p58 antibodies which needed 0.1% Triton-X100 instead of saponin for permeabilization or −20°C methanol for 20 min for fixation and permeabilization. Fibers were incubated with primary antibodies for 2 h at room temperature (or overnight at 4°C) and with secondary antibodies for 2 h at room temperature, counterstained with Hoechst 33342, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). All washes were 5–15 min at room temperature with PBS containing 0.04% saponin or with PBS alone for samples permeabilized with Triton X-100.
9
Immunofluorescent Analysis of RAGE and Rab31 in INS-1 Cells
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INS-1 cells were seeded to cell culture slides and treated with GS for the indicated time. After treatment, the cell culture slides were fixed with 4% paraformaldehyde for 20 min, followed by incubation with 0.1 M glycine/PBS solution for 5 min and incubation with 0.1 % Tween-20/PBS solution for another 5 min to permeabilize the plasma membrane. Unspecific binding of the antibody was prevented by blocking the slides with 5% calf serum and the samples were then incubated with anti-RAGE-antibody (Abcam) and anti-Rab31-antibody (Bioworld) at 4°C overnight. The slides were then incubated for 1 h with a donkey-anti-rabbit Delight 594 or a donkey-anti-mouse FITC-conjugated antibody (Bioworld). Finally, the slides were incubated with Hoechst33342 and mounted with Vectashield (Vector laboratories, Burlingame, CA). The fluorescence was visualized with an FV1200 laser scanning confocal microscope (Olympus). Images were processed with an FV10-ASW viewer (Olympus).
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