Tyrosine hydroxylase (TH) is an enzyme important for DA synthesis and is a marker of dopaminergic neurons (Fujino et al., 2020). SN coronal sections (30 μm in thickness) were cut from –4.5 to –6.2 mm caudal to bregma in the frozen brain tissue by using a cryotome (Thermo Fisher Scientific) and then were blocked in Tris-buffered saline with 3% bovine serum albumin for 2 hours and incubated with a TH primary antibody (rabbit, 1:500, Abcam, Cambridge, UK, Cat# ab6211, RRID: AB_2240393) overnight. After washing with Tris-buffered saline, the secondary antibody Alexa Fluor® 488 donkey anti-rabbit IgG (1:500, Abcam, Cat# ab150073, RRID: AB_2636877) was applied to the sections for 2 hours at room temperature. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime Biotechnology, Shanghai, China) was used to stain the nuclei for 15 minutes at room temperature, and the cells were washed with phosphate buffer solution three times. The sections were sealed with 50% glycerol, and the detection of fluorescence signals was conducted using an SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany). Results are shown as TH-positive neurons in the SN.
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride
Manufactured by Beyotime
Sourced in China
2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride is a chemical compound used for research and laboratory applications. It is a white to off-white crystalline solid.
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Lab products found in correlation
Cryotome, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Abcam (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Alexa fluor 488 labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Beyoclick edu cell proliferation kit with alexa fluor 488, by Beyotime (1 mentions)
Sp5 2 confocal microscope, by Leica (1 mentions)
Alexa fluor 488 goat anti rabbit igg h c, by Thermo Fisher Scientific (1 mentions)
Albumin bovine 5, by Solarbio (1 mentions)
Alexa fluor 568 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Abcam (1 mentions)
Sc 390811, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Alexa fluor 647 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Eclipse ti sr, by Nikon (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
10 protocols using 2 4 amidinophenyl 6 indolecarbamidine dihydrochloride
1
Immunofluorescent Quantification of Dopaminergic Neurons
2
Immunostaining of α-Synuclein Fibrils
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Cells were seeded in 8 well-plate formats. The day after, cells were treated with 10 μM concentrations of α-Syn fibrils for 24 h. At the end of the treatment, cells were extensively washed with PBS and then fixed for 15 min in 4% formaldehyde (methanol-free) in PBS. Then wash with PBS 3 × 10 min and counterstain for 5 min with the nuclear stain 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime) and wash with PBS 3 × 10 min again.
3
Immunofluorescence Staining of HeLa Cells
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HeLa cells were grown on glass coverslips for 24 h in complete medium. Remove the growth medium and wash the cells in Dulbecco’s PBS (DPBS). Fix for 15 min in 4% formaldehyde (methanol-free) in DPBS and permeabilize for 10 min with 0.1% Triton X-100 in DPBS. Wash with DPBS once and incubate for 1 h at room temperature (RT) in blocking buffer (4% FBS in DPBS) to block nonspecific sites. Incubate the cells overnight at 4 °C with primary antibodies (SERF1A polyclonal antibody, Invitrogen) in blocking buffer. Remove the primary antibodies and rinse the cells briefly in DPBS at the following day. Then wash with DPBS 3 × 10 min. Incubate for 2 h at RT with secondary antibodies (Alexa Fluor 488–labeled goat anti-mouse IgG(H + L), Beyotime) in blocking buffer. Counterstain for 5 min with the nuclear stain 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime). Wash with DPBS 3 × 10 min and sealed with antifluorescence quenching sealing tablets.
4
Immunofluorescent Localization of EtAMA1 and Interactors
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To identify co-localization of EtAMA1 and its interaction proteins, EtRON2 and EtESP, in E. tenella, the purified sporozoites were transferred to glass slides and air-dried. Sporozoites were fixed in 2% paraformaldehyde in PBS, air-dried, and permeabilized with 1% Triton X-100 in PBS for 15 min. The slides were blocked with 2% (w/v) bovine serum albumin in PBS for 2 h at 37 °C and incubated with polyclonal anti-rEtAMA1 mouse serum, and anti-rEtESP or anti-rEtRON2 rabbit serum [43 (link)] diluted in PBS (1:100) at 37 °C for 2 h. Slides were then incubated with Alexa Fluor™ 488 chicken anti-rabbit IgG or Alexa Fluor™ 647 chicken anti-mouse IgG secondary antibodies (1:500 dilution) for 1 h at 37 °C. Nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime) (10 mg/mL) for 20 min at room temperature. After each step, the slides were washed three times for 10 min each with PBS containing 0.05% Tween 20. Slides were then mounted using Fluoromount Aqueous Mounting Medium (Sigma-Aldrich) and observed under a laser scanning confocal microscope (Zeiss, Germany).
5
EdU Proliferation Assay for Cell Screening
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The EdU (5-Ethynyl-2′-deoxyuridine) incorporation assay was performed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, China), following the instructions provided in the operating manual. Briefly, the cells were seeded into 24-well plates at a density of 1 × 104 cells per well. Once the cells adhered to the plates, they were treated with various drugs and maintained at 37 °C in a 5% CO2 incubator for 2 days. Then, the test cells were incubated with 10 μM of EdU for 1 h. Subsequently, the cells were fixed with 3.7% paraformaldehyde (PFA) for 15 min, followed by washing in PBS and permeabilization with 0.3% Triton X-100. The cells were then stained with freshly made Click Additive Solution at room temperature for 30 min. Nuclei were counterstained with DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, Beyotime, China). Finally, images were captured using a fluorescence microscope.
6
Immunofluorescence Analysis of DEK and β-Tubulin
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MKN-1 gastric cancer cells grown on coverslips was fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.5% TritonX-100 for 10 min. Then washed again with PBS and blocking was performed with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for one hour at the room temperature (RT). Primary antibodies against DEK (1:50; BD Biosciences, USA) and β-Tubulin (1:50; Santa Cruz Biotechnology, USA) were incubated with cells at 4°C overnight. After more washes, cells were incubated with Alexa Fluor® 488 Goat Anti-Rabbit IgG (H + C) (A11008, 1:1000, Invitrogen, USA) and Alexa Fluor® 568 Goat Anti-Mouse IgG (H + L) (A11004, 1:1000, Invitrogen, USA) for 1 h. Subsequently, cells were washing again with PBS and counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (C1006, Beyotime, China). Coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, China) [17 (link)]. Finally, the immunofluorescence signals were visualized and recorded by Leica SP5II confocal microscope.
7
Microglial Activation Quantification Protocol
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Increased microglial activity is evidenced by a profound shift in morphology and upregulation of ionized calcium-binding adapter molecule 1 (Iba1) that can be easily visualized using immunofluorescence.20 (link) Perfused spinal cord (L4) and bilateral DRG (L4) were sectioned into 5 μm coronal sections. Three sections were sectioned per 1 tissue sample. Briefly, sections were rinsed extensively in PBS for 3 times followed by a 10-minute fixation in 4% paraformaldehyde. The tissue was then incubated in a primary antibody solution (rabbit anti-Iba 1 1:1000; wako 019-19741) in PBS for 24 hours at 4°C. After being rinsed with PBS, the tissue was incubated for 1 hour in a secondary antibody (biotinylated immunoglobulin G goat antirabbit 1:600), rinsed with PBS, and then incubated for 1 hour. After being rinsed in PBS, the tissue was then incubated in 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime, Shanghai, China) for 10 minutes. The intensity of fluorescence was analyzed using Image J (National Institutes of Health, Bethesda, MD) analysis software.
8
Immunofluorescent Staining of GCLC
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1×105 cells were seeded on glass coverslips and grew for three days. Cells were washed three times with PBS and fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 and then blocked with 5% goat serum. Sections were incubated with GCLC primary mouse antibody (1:100, sc-390811, Santa Cruz, CA, USA) overnight at 4°C. After washed with PBST for three times, the coverslips were incubated with Alexa Fluor® 488-conjugated goat anti-mouse secondary antibody (1:100, Abcam, Cambridge, UK. Cat No#ab150117) for 2 hours in dark. Neclei were counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime, Shanghai, China. Cat No#C1005). All the sections were observed under a fluorescence microscope (Olympus).
9
Immunostaining of Brain Tissue for BDNF and GFAP
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The frozen brain tissue section was washed in PBS for 15 min three times and inubated with 1% Triton in PBS for 15 min. Slides were inucbated with 5% normal donkey serum for 90 min. Thereafter, the slides were incubated with primary antibodies against BDNF and GFAP at 4°C overnight and were subsequently washed in PBS for 20 min three times. Alexa Fluor®488- and Alexa Fluor®647-conjugated donkey-anti-rabbit IgG were used to amplify the protein signaling (Jackson ImmunoResearch, West Grove, PA, USA), and the incuabtion was performed at room temperature for 60 min. To detect nuclei, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime) was incubated with the slides for 15 min. The slides were photographed with a confocal scanning microscope (Leica, Wetzlar, Germany) installed with Image J software.
10
TUNEL Assay for Apoptosis Detection in Ileum
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To detect apoptotic cell death in ileum tissues, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (In Situ Cell Death Detection Kit, Roche, Mannheim, Germany) was performed using TUNEL detection kit, according to manufacturer’s instructions (Cat. No. 11684817910, Roche, Mannheim, Germany). Ileum tissue sections were incubated with proteinase K for 20 minutes at room temperature and washed with PBS. After that, the sections were incubated in terminal deoxynucleotidyl transferase buffer containing fluorescein isothiocyanate-conjugated dUTP at 37°C for 60 minutes. Morphological changes in the nuclei were observed by counterstaining with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime, Shanghai, China). Sections were analyzed under a Nikon fluorescence microscope (Eclipse Ti-SR, Nikon Corporation, Tokyo, Japan).
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