Cells were detached and stained with violet LD (#L34955; Invitrogen, Carlsbad, CA) as a live–dead discrimination marker. Cells were then stained with Cd11b‐PE/Cy7 (1:500; #25‐0112‐82; eBioscience, San Diego, CA), washed with PBS and then permeabilized using Fix/Perm buffer (BD Biosciences) and stained with primary antibody Tubb3 (1:1,000, #ab18207; Abcam) followed by secondary antibody D649 (1:800, #406406; Biolegend, San Diego, CA). Isotype controls (rabbit IgG, #171870; Abcam; rat IgG, #25‐4031‐82; eBioscience) were used to account for background staining. Cells were fixed using 4% paraformaldehyde and analyzed using a CyAN cytometer. FlowJo (Treestar, OR) was used to analyze flow cytometry data.
Tubb3
Manufactured by Abcam
Sourced in United Kingdom
TUBB3 is a protein that is a component of microtubules, which are structural elements within cells. It is involved in the formation and function of microtubules.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b pe cy7, by Thermo Fisher Scientific (2 mentions)
Fix perm buffer, by BD (2 mentions)
Violet ld, by Thermo Fisher Scientific (2 mentions)
Huc d, by Thermo Fisher Scientific (1 mentions)
Phospho histone h3, by Merck Group (1 mentions)
Lsm 800 confocal microscope, by Adobe (1 mentions)
Hnk 1, by Developmental Studies Hybridoma Bank (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Clic4, by Abcam (1 mentions)
Fam49b, by Abcam (1 mentions)
Horseradish peroxidase conjugated igg secondary antibody, by Cell Signaling Technology (1 mentions)
Becn1, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
Cryostat, by Thermo Fisher Scientific (1 mentions)
Rhodopsin, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Calretinin, by Merck Group (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
10 protocols using tubb3
1
Multiparametric Flow Cytometry Analysis
2
Analysis of Cell Viability and Tubulin-3 Expression
3
Immunohistochemical Analysis of Neural Markers
4
Immunofluorescence Analysis of Neurite Outgrowth
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Following different interventions for 24 h, the F11 cells were fixed by 4% paraformaldehyde and permeabilized by 0.1% TritonX-100 (Sigma Aldrich) for 10 min. Unspecific bindings were blocked using 8% bovine albumin at room temperature for 1 hour. Cells were incubated with primary antibody TUBB3 (1:100, Abcam) overnight at 4°C. The next day, the cells were incubated with goat anti-mouse antibody (Alexa Fluor 488, 1:500, Abcam). DAPI was used to visualize cell nucleus. The cells were detected under the Olympus FV1200 confocal microscope (Olympus). The number and length of neurites were calculated by Image J from five separated high-power fields in each group. Each experiment was performed three times.
5
Exosomal Protein Validation by WB
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The proteomic results from HPLC/MS were confirmed by western blotting. Samples of 5 µg of total exosomal proteins from both the cancer group and the control group were used for testing. The following antibodies were used for western blotting: CLIC4 (Abcam, No183043, UK), ATK1 (Abcam, No89402, UK), EPAP II (Abcam, No188320, UK), SNX3 (Abcam, No56078, UK), FAM49B (Abcam, No121299, UK), KIND3 (Abcam, No68040, UK), LTF (Abcam, No10110, UK) and TUBB3 (Abcam, No52623, UK).
6
Protein Expression Analysis of Differentiated hNSCs
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Total proteins were extracted from the differentiated hNSCs using RIPA lysis buffer with 1 mM phenylmethanesulfonylfluoride. A total of 20 μg of protein samples was separated by a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. Each membrane was blocked for 1 h with 5% defatted milk powder at room temperature and then incubated with TUBB3 (1:1000, #ab53623, Abcam, Cambridge, UK), ATG5 (1:1000, #12994, Cell Signaling Technology, Danvers, MA, USA), BECN1 (1:1000, #3495, Cell Signaling Technology, Danvers, MA, USA), LC3A/B (1:1000, #12741, Cell Signaling Technology, Danvers, MA, USA), CTNNB (1:1000, #8480, Cell Signaling Technology, Danvers, MA, USA), MYC (1:1000, #5605, Cell Signaling Technology, Danvers, MA, USA) or ACTB (1:5000, #AB2001, ABways, Shanghai, China) primary antibody at 4 °C overnight. The membrane was then incubated with corresponding horseradish peroxidase-conjugated IgG secondary antibodies (1:10,000, #7074P2, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The blot images were captured by the gel Bio-rad imager. The gray value of each band was measured using ImageJ. The relative expression of each band was calculated by comparing the target protein band with GAPDH. Each group had three independent samples.
7
Immunohistochemical Analysis of Retinal Tissues
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The eyes of mice or rats were removed, punctured with a fine gauge needle, and placed in 4% PFA at 4°C for 12 h. Eyes were then cryoprotected in 30% sucrose for 12 h and embedded in OCT medium (Sakura Finetek). Ten‐micrometer tissue sections were cut at −20°C in a cryostat (Thermo Scientific) and collected on the poly‐L‐lysine coated slides. For immunohistochemistry, sections were permeabilized in PBS with 0.2% Triton X‐100 for 20 min and then blocked in PBS with 10% bovine serum albumin (BSA) for 1 h. Retinal sections were incubated with the primary antibodies, including GFAP (1:200, Abcam), GS (1:200, Abcam), NeuN (1:100, Abcam), TUBB3 (1:100, Abcam), calretinin (1:500, Chemicon), calbindin (1:200, Abcam), rhodopsin (1:1,000, Sigma), protein kinase Cα (PKCα, 1:200, Abcam), nestin (1:100, Santa Cruz), or vimentin (1:100, Santa Cruz) at 4°C for 24 h. The sections were washed with PBS and then incubated with FITC‐ or Cy3‐conjugated secondary antibody (1:500, Invitrogen) overnight at 4°C. Slides were finally mounted and observed using an Olympus IX‐73 microscope.
8
Quantification of Cellular and Exosomal Proteins
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Total protein of cells (1×106) or exosomes (1 μg/ml) were isolated using the cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The procedure of western blots was performed as previously described [58 (link)]. The primary antibodies against E-cadherin (1:1000), N-cadherin (1:1000), vimentin (1:500), fibronectin (1:1000), CD9 (1:1000), CD63 (1:1000), GM130 (1:1000), TUBB3 (1:1000), PPP2R1B (1:1000), and GAPDH (1:5000) were obtained from Abcam (Abcam Trading Company Ltd., Shanghai, China). The intensity of protein bands was determined via Imagej software [64 (link)].
9
Protein Isolation and Western Blot Analysis
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Proteins were isolated using lysis solution (62.5 mM Tris, pH 6.8, 100 mM DTT and 2% SDS, 10% glycerol, traces of bromphenol blue) and benzonase (Pure Grade, Merck, Darmstadt, Germany) as previously described [45 (link)]. Isolated proteins were separated on a 7% to 15% polyacrylamide gradient SDS-PAGE gel and then transferred to a PVDF membrane (Immobilon P; Millipore, Billerica, MA, USA). Membranes were first incubated with primary antibodies against MYC (Novus Biologicals, Abingdon, UK), TUBB3 (Abcam, Cambridge, UK), NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA), MCL1 (Proteintech, Rosemont, IL, USA), BAX (Cell Signaling Technology, Danvers, MA, USA), ACTB (Abcam, Cambridge, UK) and then with goat anti-rabbit or rabbit anti-goat horseradish peroxidase-linked secondary antibodies (Agilent Dako, Santa Clara, CA, USA). Protein detection was performed with Clarity Western ECL Substrate (BioRad, Hercules, CA, USA) according to manufacturer’s instructions on ChemiDoc XRS+ System (BioRad, Hercules, CA, USA). Protein band intensity was evaluated densitometrically using ImageJ 1.45S software, and final values were calculated by dividing the test proteins by ACTB and then normalizing obtained values by the control protein.
10
Immunofluorescence Staining of Neural Precursors
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Cells and neurospheres were washed with PBS and fixed in 4% paraformaldehyde for 10 min at 37 °C, followed by treatment with absolute methanol for 10 min at 4 °C and blocking in PBS containing 1% FBS for 10 min. The samples were then incubated with primary antibodies at 37 °C overnight, rinsed twice with PBS, and incubated with the secondary antibodies for 1 h. The samples were observed under a confocal microscope LSM700 (Carl Zwiss, Thornwood, NY). Primary antibodies were as follows: anti-nestin (Covance), anti-N-FM (Millipore), and TUBB3 (Abcam). Secondary antibodies were as follows: Alexa 594-conjugated donkey anti-mouse IgG (Molecular Probes, Eugene, OR), Alexa 594-conjugated donkey anti-rabbit IgG (Molecular Probes), and R-PE-conjugated rabbit anti-rat IgG (Southern Biotechnology Associates, Inc., Birmingham, AL).
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