Nerve cords were fixed for one hour at 4°C in 4% paraformaldehyde just after dissection. The 5′ biotin-labeled specific antisense probe and sense probe (negative control) were generated from the 842–1479 nucleotides sequence of hmcalr mRNA (Genbank Accession Number KF709537). After PCR amplification and the insertion of the product in pGEM-T easy vector system (Promega, USA), the RNA sequence of interest was obtained by in vitro transcription using DIG/Biotin RNA-labeling kit according to the manufacturer’s instructions (Roche, Switzerland). The hybridization protocol was performed as previously described [30 (link)]. Nerve cords were incubated with a secondary anti-biotin antibody conjugated to Alexa Fluor 488 (dilution 1: 5000 in PBS) (Invitrogen, USA). Samples were rinsed with PBS. Prior to mounting with Glycergel (Sigma Life Science, USA), the cell nuclei were counterstained by Hoechst 33342 fluorescent dye (1:1,000, Invitrogen USA) for 10 min. Slides were kept at 4°C in the dark until observation with a Zeiss LSM700 confocal microscope.
Hoechst 33342 fluorescent dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33342 is a fluorescent dye used for DNA staining. It binds to the minor groove of double-stranded DNA, emitting a blue fluorescence upon excitation. This property makes Hoechst 33342 a useful tool for various applications in cell and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Sodium bicarbonate, by Thermo Fisher Scientific (3 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab21286, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
Ab6640, by Abcam (2 mentions)
Ep798y, by Abcam (2 mentions)
Ab756, by Merck Group (2 mentions)
Sc 32720, by Santa Cruz Biotechnology (2 mentions)
Sc 167530, by Santa Cruz Biotechnology (2 mentions)
Sc 9068, by Santa Cruz Biotechnology (2 mentions)
Fitc or texas red labeled secondary antibody, by Vector Laboratories (2 mentions)
Ab5694, by Abcam (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fluorescent daunorubicin hcl, by Selleck Chemicals (2 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (2 mentions)
Nilotinib, by LC Laboratories (2 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Crystal violet, by Merck Group (1 mentions)
13 protocols using hoechst 33342 fluorescent dye
1
In Situ Hybridization of hmcalr mRNA
2
Evaluation of Anticancer Agent Efficacy
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Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum (FBS) and 0.25% trypsin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 5-Fu was purchased from NanJing KeyGen Biotech Co., Ltd. (Nanjing, Jiangsu, China). The anti-human ABC sub-family G member 2 (ABCG2) antibody (catalog no. 3765-1) was purchased from Epitomics (Burlingame, CA, USA). The anti-human B-cell lymphoma (BCL)-2 (catalog no. 15071) and anti-human BCL-2-associated X protein (catalog no. 2774) antibodies were purchased from CST Biological Reagents Company Limited (Shanghai, China). The anti-human forkhead box protein M1 (FOXM1; catalog no. ab55006) antibody was purchased from Abcam (Cambridge, MA, USA). The anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; catalog no. G8795) antibody was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Hoechst 33342 fluorescent dye and verapamil were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Matrigel invasion chambers were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Crystal violet was purchased from Sigma-Aldrich (Merck Millipore).
3
Comprehensive Immunohistochemistry Protocol for Skin Tissue
4
BrdU Proliferation Assay for U87 and U251 Cells
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5-Bromo-20-deoxyuridine (BrdU) incorporation kit (cat #11,296,736,001 Roche, IN, USA) was used according to the manufacturer’s recommendations to evaluate the proliferative capacity of the cell lines under the effect of the different treatments. 5 × 103 U87 cells and 4 × 103 U251 cells were grown in 24-well glass slides and maintained as previously described. After 72 hours of treatment, cells were incubated with BrdU labeling medium for 50 min. After cell fixation, the incorporation of BrdU was detected by immunofluorescence using a fluorescence-labeled secondary antibody. Cell nuclei were stained with Hoechst 33,342 fluorescent dye (1 mg/mL) (Thermo Scientific, USA). Positive BrdU cells were visualized in an Olympus Bx43F microscope (Olympus, PA, USA). Cell counting was done with the ImageJ software 1.45S (National Institutes of Health, USA), and the percentage of BrdU-positive cells was calculated considering the nuclei stained with Hoechst as the total number of cells.
5
Antibacterial Activity Evaluation Protocol
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Pure cultures of Str. pyogenes, S. aureus, S. epidermidis, P. aeruginosa, and Ent. Faecium were used for the study of antibacterial activity using the method adapted from Koller M. et al. [31 (link)].
Before starting, standard solutions containing 2 × 106 CFU/mL each were prepared in RPMI-1640 medium (Thermo Fisher Scientific, USA) with the addition of 0.3 g/L L-glutamine and 2 g/L sodium bicarbonate (Thermo Fisher Scientific, USA), and samples were placed in wells of a 6-well plate. Fifty microliters of the bacterial suspension was applied as a drop to each test sample and incubated in a humid thermostat at 37 °C (Figure 1 ). The next day, a drop of bacterial suspension from the sample was transferred to a Petri dish with appropriate culture medium and cultured further for 24 h to evaluate bacterial growth. Bacterial colonies were stained with Hoechst 33342 fluorescent dye (Thermo Fisher Scientific, USA) and were counted using an Axioskop 40FL microscope (Carl Zeiss, Jena, Germany). Measurements in each experimental group were performed in three repetitions.
Before starting, standard solutions containing 2 × 106 CFU/mL each were prepared in RPMI-1640 medium (Thermo Fisher Scientific, USA) with the addition of 0.3 g/L L-glutamine and 2 g/L sodium bicarbonate (Thermo Fisher Scientific, USA), and samples were placed in wells of a 6-well plate. Fifty microliters of the bacterial suspension was applied as a drop to each test sample and incubated in a humid thermostat at 37 °C (
6
Histological and Immunofluorescence Analysis
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Paraffin-embedded formalin-fixed samples were cut into 5 μm sections. After dewaxing and rehydration, sections were stained with haematoxylin, phloxin and saffron (HPS) for routine histological analysis.
Immunofluorescence staining of paraffin sections was performed to detect Ki67, integrin alpha-6, type VII and XVII collagen. 5-μm paraffin-embedded sections were deparaffinized and rehydrated. After heat unmasking, the sections were incubated with a solution of 5% BSA to block nonspecific binding. Sections were then incubated overnight with the primary antibodies at 4°C overnight, followed by an AlexaFluor-568 secondary anti-mouse or anti-rabbit antibodies (Molecular Probes, Invitrogen) for 1 h at room temperature. Nuclear counterstaining using Hoechst 33342 fluorescent dye (Thermo Fisher Scientific, MA, United States) was carried out routinely.
Immunofluorescence staining of paraffin sections was performed to detect Ki67, integrin alpha-6, type VII and XVII collagen. 5-μm paraffin-embedded sections were deparaffinized and rehydrated. After heat unmasking, the sections were incubated with a solution of 5% BSA to block nonspecific binding. Sections were then incubated overnight with the primary antibodies at 4°C overnight, followed by an AlexaFluor-568 secondary anti-mouse or anti-rabbit antibodies (Molecular Probes, Invitrogen) for 1 h at room temperature. Nuclear counterstaining using Hoechst 33342 fluorescent dye (Thermo Fisher Scientific, MA, United States) was carried out routinely.
7
Comprehensive Immunohistochemistry Protocol for Skin Tissue
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Paraffin or frozen 5 µm sections of skin tissue samples were stained with the following antibodies: CD31 antibody (1:100, ab28364 Abcam) for detection of endothelial cells; Calponin (1:100, EP798Y, Abcam) and α-SMA (1:200, ab5694, Abcam) for detection of perivascular cells, and F4/80 (1:100, ab6640, Abcam) for assessing macrophages in vivo. Collagen type I (1:200, ab21286, Abcam), collagen type IV (1:100, AB756, Chemicon), collagen type XVIII (1:100, sc-32720, Santa Cruz), collagen type VI (1:50, sc-167530, Santa Cruz), and fibronectin (1:500, sc-9068, Santa Cruz) were used for extracellular matrix and basement membrane evaluation. Incubation with primary antibody was followed by FITC or Texas Red labeled secondary antibody (1:500, Vector Laboratories). Nuclei were stained with Hoechst 33342 fluorescent dye (Thermo Scientific).
8
Analyzing Nuclear Morphology in Apoptosis
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To assess nuclear morphology changes associated with apoptosis, the cells were grown onto glass slides, the culture media were removed, and the cells were fixed in 3.7% buffered formaldehyde for 10 min at room temperature. After incubation in phosphate buffered saline (PBS) for 10 min, the slides were washed with deionised water and stained with Hoechst 33342 fluorescent dye (Thermo Fisher Scientific Inc. Rockford, IL, USA) diluted at 1 μg/mL for 15 min at room temperature. The slides were washed and mounted. The cells were analyzed using a Fluorescence Microscope Axiostar Plus (Karl Zeiss Microscopy GmbH, Jena, Germany).
9
Live-cell Imaging of MESC Migration
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A total of 5 × 104 MESCs were seeded into the wells of a 6-well plate (polystyrene and dECM-coated) in complete growth medium with the addition of 1 μg/mL Hoechst 33,342 fluorescent dye (Thermo Fisher Scientific, Waltham, MA, USA). Then, the plate was cultivated for 24 h at 37 °C and 5% CO2 in the CQ1 Confocal Imaging Cytometer (Yokogawa Electric Corp., Tokyo, Japan). Twenty fields of view were photographed every 15 min. Time-lapse series of merged phase-contrast and fluorescent images were analyzed with Manual Tracking ImageJ plugin in order to obtain individual tracks of 50 cells. Mean cell speed and directionality score were calculated using Chemotaxis and Migration Tool software Version 2.0 (Ibidi GmbH, Martinsried, Germany).
10
BrdU Assay for Cellular Proliferation
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5-bromo-2′-deoxy-uridine (BrdU) incorporation assay was used to test the proliferative effect of PIBF. 4 × 103 U87 cells were seeded in chambered cell culture slides and maintained as described in the “Cell Culture” section. After 24 h, the medium was replaced for phenol red and hormone-free DMEM medium. Cells were treated for 4 days as described in “Treatments” section. After treatments, the bromo-2′-deoxy-uridine Labeling and Detection Kit I (Roche, BW, DE) was used according to the manufacturer's protocol. Hoechst 33342 Fluorescent Dye (Thermo Scientific, MA, USA) was used to stain the DNA. The fluorescence signal was observed at dual-wave lengths of 486 and 515–565 nm in the fluorescence microscope Olympus Bx43F (Olympus, TYO, JPN). The number of cells with BrdU incorporation was obtained using the ImageJ software (National Institute of Health, WA, USA) and the percentage of cells positive for BrdU was calculated considering the total number of cells stained with Hoechst.
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