The following antibodies were used in these studies: mouse anti-aSyn (#610787, BD Biosciences, San Jose, CA); rabbit anti-ENSA (#D5Z1U), rabbit anti-β3-tubulin (#D65A4), and AP-linked anti-mouse and anti-rabbit IgG (Cell Signaling Technology, Danvers, MA); chicken anti-MAP2 (#CPCA-MAP2, EnCor Biotechnology, Gainesville, FL); rabbit anti-TH (#AB152, Millipore, Billerica, MA); mouse anti-β-actin (#A5316 or #A5441, Sigma-Aldrich); and anti-rabbit IgG-Alexa Fluor 488 and anti-chicken IgG-Alexa Fluor 594 (Invitrogen, Carlsbad, CA).
Rabbit anti β3 tubulin
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-β3-tubulin is a primary antibody that specifically binds to the β3-tubulin protein. It is used in research applications to detect and localize the β3-tubulin protein, which is a component of microtubules.
Automatically generated - may contain errors
Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti th, by Merck Group (1 mentions)
Anti chicken igg alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Mouse anti asyn, by BD (1 mentions)
Mouse anti map2, by Merck Group (1 mentions)
Tcs sp5x, by Leica (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Ab223052, by Abcam (1 mentions)
Ab126729, by Abcam (1 mentions)
Ab169532, by Abcam (1 mentions)
Ab88743, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Pm036, by Progen Biotechnik (1 mentions)
Gp62 c, by Progen Biotechnik (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti inos, by Cell Signaling Technology (1 mentions)
Rabbit anti f4 80, by Abcam (1 mentions)
Rabbit anti gfap, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit anti β3 tubulin
1
Immunofluorescence Staining of Neural Markers
2
Immunofluorescence Analysis of Neuronal Markers
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Early NPCs were generated and Ssrp1 levels were knocked down as described above. The cells were fixed with 100% ethanol for 10 min and processed for immunofluorescence. Permeabilization and blocking was performed for 1 h at room temperature with 1% BSA and 0.1% NP-40 in PBS. Incubation with primary antibodies was carried at room temperature for 2 h by using rabbit anti-β3-tubulin (1:300; Cell Signaling) and mouse anti-MAP2 (1:300; Millipore). After washing in blocking buffer, the secondary antibodies anti-rabbit and anti-mouse Alexa Fluor 568 (1:1,000; Life Technologies) were applied for 2 h at room temperature. Slides were extensively washed in PBS and nuclei were counterstained with DAPI before mounting. Fluorescence images were acquired using a laser scanning confocal microscope (TCS SP5-X; Leica), equipped with a white light laser, a 405-diode UV laser, and a 40× objective lens.
3
Protein Expression Analysis by Western Blotting
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Total protein extraction was performed using a radioimmunoprecipitation assay solution. Protein concentration was measured using the BCA assay, and an equal quantity of each sample (100 μg) was used for Western blotting. Following SDS-PAGE, the proteins were transferred onto 0.45 µm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). Membranes were blocked with 5% (w/v) non-fat dry milk in Tris base saline Tween 20 (TBST; 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween-20 (v/v)) for 1.5 hours, and then, washed thrice for 10 minutes in TBST, followed by incubation at 4 °C overnight with mouse anti-Alix (ab88743, Abcam), rabbit anti-CD9 (ab223052, Abcam), rabbit anti-Netrin1 (ab126729, Abcam), mouse anti-NF (#2836, Cell Signaling Technology), rabbit anti-microtubule-associated protein (MAP2) (#4542, Cell Signaling Technology), rabbit anti-β3-tubulin (#5568, Cell Signaling Technology), rabbit anti-Phox2b (25276-1-AP, Proteintech Group Inc, USA), rabbit anti-Hand2 (abs117611, Absin Bioscience Inc., China), GAPDH (10494-1-AP, Proteintech Group Inc), and Lamin A/C (ab169532, Abcam). Next, the membranes were washed thrice in TBST and incubated with a secondary antibody at room temperature for 2 hours. Following washing with TBST, the reaction was detected using chemiluminescence and the Western Bright ECL detection kit (WBKLS00100, USA).
4
Immunofluorescence Microscopy Protocol for SH-Sy5y Cells
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40 x 104 SH-Sy5y cells, were seeded in 24-well culture plates on coverslips. Cells were treated as specified for the specific experiments. Cells were fixed with 4% formaldehyde (VWR 9713.1000) for 15–20 min at RT, and permeabilized with methanol for 10 min at −20°C and blocked in PBS 3% donkey serum for 1 h at RT. The fixed cells were stained with rabbit anti-LC3 (Nordic Biosite, PM036, dilution 1:2000), guinea pig anti-p62 (Progen, GP62-C, dilution 1:200), rabbit anti-β3-tubulin (Cell Signaling, 2146, 1:100) for 1 h at RT. The cells were washed three times with PBS and incubated for 1 h with a secondary antibody of donkey anti-rabbit Alexa 488 antibody (Thermo Fischer, A-21206, dilution 1:1000) for LC3, a secondary goat anti-guinea pig Alexa A568 (Thermo Fischer, A-11075) for P62 and DAPI. Antibodies were diluted in PBS 1% donkey serum. After three washes with PBS, coverslips were mounted using Prolong Gold mounting medium (Thermo Fischer P36934). Images were acquired on a Zeiss LSM 800 confocal microscope and processed with the Fiji software package. LC3 foci were counted manually in 25 randomly selected cells per condition. HSV1 capsids associated with the nuclear rim were quantified over 100 nuclei.
5
Cell Uptake and Immunofluorescence Staining
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For the cell uptake experiment, a medium containing NGF, EVs−NGF, and EVs−cl−NGF (FITC-labeled NGF, 5 μg/mL) was added to the Transwell™ coculture model for 6 h. Cells were removed from the incubator, and the cells were fixed with immune tissue fixative. Finally, the cell membrane was labeled with phalloidin 561, and the cell nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI).
For immunofluorescent dual-labeling staining, cells or frozen sections were fixed with immune tissue fixative and incubated with Triton X-100 (0.3%) followed by goat serum (10%) for 2 h. The cells were incubated with the first antibody overnight at 4 °C, washed, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (A-11034/A-11005, 1:250, Thermo Fisher Science) for 2 h at room temperature. Next, the cell nucleus was stained with DAPI. The primary antibodies were as follows: rabbit anti-F4/80 (1:200, Abcam, Cambridge, UK), rabbit anti-cleaved-Caspase3 (c-caspase3) (1:1000, Abcam, Cambridge, UK), mouse anti-ARG-1 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-iNOS (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-NurN (1:50, Cell Signaling Technology, MA, USA), rabbit anti-GFAP (1:200, Cell Signaling Technology, MA, USA), and rabbit anti-β3-Tubulin (1:200, Cell Signaling Technology, MA, USA).
For immunofluorescent dual-labeling staining, cells or frozen sections were fixed with immune tissue fixative and incubated with Triton X-100 (0.3%) followed by goat serum (10%) for 2 h. The cells were incubated with the first antibody overnight at 4 °C, washed, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (A-11034/A-11005, 1:250, Thermo Fisher Science) for 2 h at room temperature. Next, the cell nucleus was stained with DAPI. The primary antibodies were as follows: rabbit anti-F4/80 (1:200, Abcam, Cambridge, UK), rabbit anti-cleaved-Caspase3 (c-caspase3) (1:1000, Abcam, Cambridge, UK), mouse anti-ARG-1 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-iNOS (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-NurN (1:50, Cell Signaling Technology, MA, USA), rabbit anti-GFAP (1:200, Cell Signaling Technology, MA, USA), and rabbit anti-β3-Tubulin (1:200, Cell Signaling Technology, MA, USA).
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