4 × 105 HCECs were seeded onto a six-well plate and transfected with 50 nM SUV39H1 or NC. Transfected HCECs were collected to perform FACS with Intracellular Flow Cytometry Kit (Cell Signaling Technology) according to the manufacturer's protocol. Briefly, HCECs were fixed with 4% formaldehyde for 15 min at room temperature. After washing with PBS, fixed HCECs were stored at − 20 °C in 90% methanol overnight. Then, HCECs were incubated with antibodies of phospho-Rb (Alexa Fluor® 488 Conjugate, 4277, Cell Signaling Technology) or CDK6 (Alexa Fluor® 488 Conjugate, ab198944, Abcam) or IgG (Alexa Fluor® 488 Conjugate, ab150077, Cell Signaling Technology) for 1 h. Furthermore, HCECs were resuspended in PBS after washing and analyzed with the Accuri™ C6 Plus (Becton Dickinson). The FlowJo software (Tree Star Inc., Ashland, OR, USA) was used to analyze the data.
Alexa fluor 488 conjugate
Manufactured by Cell Signaling Technology
Sourced in United States
The Alexa Fluor® 488 Conjugate is a fluorescent dye that can be used to label and detect various biomolecules, such as proteins, nucleic acids, and small molecules. It has an excitation wavelength of approximately 488 nm and an emission wavelength of approximately 520 nm, making it suitable for use in applications that utilize blue or green fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugate, by Cell Signaling Technology (3 mentions)
Alexa fluor 647 conjugate, by Cell Signaling Technology (3 mentions)
Facscalibur, by BD (3 mentions)
Alexa fluor 555 conjugate, by Cell Signaling Technology (2 mentions)
Cy3 affinitypure donkey anti guineapig, by Jackson ImmunoResearch (2 mentions)
Alexa fluor plus 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 affinity pure conjugate, by Jackson ImmunoResearch (2 mentions)
Igg xp isotype control alexa fluor 488 conjugate, by Cell Signaling Technology (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Facscalibur flow cytometry, by BD (2 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Application suite x las x, by Leica (1 mentions)
Rabbit anti lc3 antibody, by Novus Biologicals (1 mentions)
Improved citrate antigen retrieval solution, by Beyotime (1 mentions)
Tcs sp5, by Leica (1 mentions)
Riluzole hydrochloride, by Bio-Techne (1 mentions)
Bay36 7620, by Bio-Techne (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
59 protocols using alexa fluor 488 conjugate
1
FACS Analysis of Epigenetic Regulator SUV39H1
2
Phenotypic Characterization of hBMSCs
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Cells were suspended in FACS buffer at a concentration of 5 × 106/ml. For surface marker staining, cells were incubated with various combinations of fluorochrome-labeled antibodies (CD90, CD44, CD105, CD73 along with isotype controls) according to manufacturer’s instructions (BD Stemflow™). For intracellular staining, cells were washed with Cytofix/Cytoperm Plus® (BD Biosciences) and stained for transcriptional factors OCT-4 (clone C30A3, rabbit mAb Alexa Fluor 488 Conjugate, Cell Signaling Technology, 5177S), SOX-2 (clone D6D9, XP® rabbit mAb Alexa Fluor 488 Conjugate, Cell Signaling Technology, 5046S), SSEA-4 (clone MC813-70, mouse mAb PE conjugate, BD Biosciences, 560128) and Nanog (clone D73G4, XP® rabbit mAb Alexa Fluor 647 Conjugate, Cell Signaling Technology, 5448S) for 30 minutes at 4 °C. Cells were analysed using a FACSCanto® cytometer (BD Biosciences). Median Fluorescence Intensity (MFI) of hBMSCs was calculated using FlowJo® software (TreeStar Inc.) and fold increase over the appropriate isotype control was averaged.
3
Immunohistochemical Analysis of Rat Tibialis Anterior Muscle
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The rats were euthanized and the tibialis anterior muscles were obtained. Then the tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The fixed tissue (5 μm) on the slides were de-paraffinized in xylene and rehydrated by graded alcohols, followed by antigen retrieval, which used citrate unmasking buffer at 95 °C, 30 min, then 115 °C, 30 s. After cooling down to RT, endogenous peroxidase activity was quenched by 3% H2O2 for 10 min. Subsequently, the slides were rinsed three times with distilled water, and transferred to PBS. The slides were then blocked with 5% goat serum in PBS for 1 h and incubated with primary antibodies for 1 h. Following extensive washing in PBS, corresponding fluorescent secondary antibodies were added for 1 h. Primary antibodies include mouse anti-SV2A (Santa Cruz Biotechnology, sc-376234, 1:100) and rabbit anti-C5b polyclonal antibody (ABclonal, A8104, 1:100), while secondary antibodies contain anti-mouse IgG (H + L) F(ab′)2 fragment (Alexa Fluor® 488 Conjugate) (Cell Signaling Technology, 4408, 1:500) and anti-rabbit IgG (H + L) F(ab′)2 fragment (Alexa Fluor® 488 Conjugate) (Cell Signaling Technology, 4412, 1:500).
4
Visualizing Autophagy in Astrocytes
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After treatment with GT, astrocytes were washed with 1X PBS and then fixed with 100% methanol at −20°C for 15 min. After fixation, they were rinsed three times in 1X PBS for 5 minutes each and blocked using normal goat serum (5%) comprising Triton X-100 (0.3%) and 1X PBS at 60 min. Subsequently, the cells were coincubated in mouse mAb anti-Glial fibrillary acidic protein (GFAP) antibody (1:50) (Alexa Fluor 555 Conjugate, Cell Signaling Technology, Beverly, MA, USA) and LC3II (Alexa Fluor 488 Conjugate, Cell Signaling Technology, Beverly, MA, USA) (1:50) antibodies at 4°C overnight in 1X PBS, 1% BSA, and 0.3% Triton X100. After incubation, the cells were washed with 1X PBS, and then nuclei were stained with DAPI. LC3II puncta were visualized and captured using a confocal microscope Leica Application Suite X (LAS X) (Leica Microsystems, Germany).
5
Immunofluorescence Staining of Autophagy Markers
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For in vitro experiments, after treatment, HepG2 cells were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature, washed twice in PBS, and blocked for 45 min at room temperature in PBS containing 6% BSA and 0.25% Triton X-100. Cells were then stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) and FITC-labeled secondary antibody (1:2000 dilution, KPL).
For in vivo experiments, paraffin-embedded tissue sections were treated with improved citrate antigen retrieval solution (Beyotime) for 30 min at 100 °C. Following twice wash with PBS, they were treated with 0.25% Triton X-100 for 15 min and blocked with PBS containing 6% BSA for 45 min at room temperature. Then, tissue sections were stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) or rabbit Atg13 antibody (1:200 dilution, Cell Signaling, 13468S) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) or Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 594 Conjugate) secondary antibody (1:300 dilution, Cell Signaling, 4412S/8889S).
All stained samples were visualized under a laser confocal microscope (Leica TCS SP5).
For in vivo experiments, paraffin-embedded tissue sections were treated with improved citrate antigen retrieval solution (Beyotime) for 30 min at 100 °C. Following twice wash with PBS, they were treated with 0.25% Triton X-100 for 15 min and blocked with PBS containing 6% BSA for 45 min at room temperature. Then, tissue sections were stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) or rabbit Atg13 antibody (1:200 dilution, Cell Signaling, 13468S) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) or Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 594 Conjugate) secondary antibody (1:300 dilution, Cell Signaling, 4412S/8889S).
All stained samples were visualized under a laser confocal microscope (Leica TCS SP5).
6
Molecular Mechanisms of GRM1 Regulation
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Riluzole hydrochloride (Tocris Bioscience, Bristol, UK) and BAY 36-7620 (Tocris Bioscience, Bristol, UK) were dissolved in DMSO. GRM1 primary antibody is specific to the C-terminal region of human GRM1α and was used at a dilution of 1:2000 for Western blotting (catalog number 36350002; Novus Biologicals, Littleton, CO). Additional primary antibodies for Western blotting or immunofluorescence include monoclonal anti-ß-actin clone AC-15 (Sigma Aldrich, St. Louis, MO); phospho-cdc2 (Tyr15), cyclin B1, phospho-histone H2AX (Ser139) (Cell Signaling, Danvers, MA). Primary antibodies for flow cytometry include phospho-histone H3 (Ser10) rabbit monoclonal (Alexa Fluor® 488 conjugate), histone H3 rabbit monoclonal (Alexa Fluor® 647 conjugate), rabbit monoclonal IgG isotype control (Alexa Fluor® 488 conjugate), rabbit monoclonal IgG isotype control (Alexa Fluor® 647 conjugate) (Cell Signaling, Danvers, MA).
7
Phospho-Histone H2A.X Immunofluorescence Assay
8
Immunofluorescence Assay for Characterizing CAFs
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In the immunofluorescence experiments, NFs and subsequently transformed CAFs were plated in six-well chamber slides (Nunc™, Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. An immunofluorescence experiment was carried out using a previously established protocol according to vendor’s instructions. Primary antibodies were added and incubated at room temperature for 1 h. The primary antibody used was α-SMA (1:100, cat no. 48938; Cell Signaling Technologies). Matched secondary antibodies were anti-mouse immunoglobulin G (IgG) (H+L), F(ab’)2 fragment (1:800, AlexaFluor 488 conjugate, cat no. 4408; Cell Signaling Technologies, Taipei, Taiwan) and anti-rabbit IgG (1:600, AlexaFluor 555 conjugated, cat no. 4413). Stained cells were mounted using Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) to counterstain the DNA. Cells were imaged with a Zeiss Axiophot (Carl Zeiss) fluorescence microscope. Microphotographs were captured using an AxioCam MRc digital video camera and analyzed using AxioVision Zeiss software (Carl Zeiss, Oberkochen, Germany).
9
Immunoblotting for Cell Signaling
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Cell lysates (20–40 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes using primary antibodies for ASPH (FB50, homemade); MMP14 (#13130S), SRC (#2109S), SRCY416 (#6943S), SRCY527 (#2105S), FAK (#13009S), Phospho-FAK Tyr397 (#8556S), FAK Tyr566/567 (#3281S), FAK Tyr925 (#3284S), and Alexa Fluor® 488 Conjugate (#5198S) from Cell Signaling Technology; MMP1 (sc-58377), ADAM12 (sc-25579), and ADAM15 (sc-16530) from Santa Cruz Biotechnology. Protein bands were visualized by ChemiDoc™ Touch Imaging System (Bio-Rad).
10
Multicolor Pancreatic Endocrine Cell Identification
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