Briefly, brain slices and N27 cells were incubated in a blocking solution (2% FBS and 0.3% Triton X-100 in PBS) for 2 h at room temperature. Brain slices were incubated with the primary antibodies in a blocking solution for 2 overnights (mouse anti-CtBP1,1:1000, BD Biosciences, cat no. 612042; mouse anti-CtBP2, 1:1000, BD Biosciences, cat no. 612044; rat anti-CD11b, 1:1000, Serotec; rabbit anti-GFAP, 1:200, DAKO; rabbit anti-NeuN, 1:500, Cell Signaling; rabbit anti-TH, 1:1000, Santa Cruz Biotechnology), while N27 cells were incubated for 1 overnight (mouse anti-CtBP1 and mouse anti-CtBP2, 1:200) at 4 °C. Afterward, tissue and cells were incubated for 2 h at room temperature with the following appropriated secondary antibodies: Alexa Fluor 594 donkey anti-mouse (Abcam), Alexa Fluor 488 donkey anti-rat (Life Technologies) and Alexa Fluor 647 donkey anti-rabbit (Life Technologies) (1:1000 for tissues and 1:200 for cells). Lastly, sections were rinsed with PBS and mounted in Fluoroshield Mounting Medium (Abcam). Images were acquired under the magnification of 40 × using a Zeiss inverted confocal microscopy (Axiobserver Z1, Zeiss).
Rabbit anti th
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-TH is a primary antibody that specifically recognizes the tyrosine hydroxylase (TH) protein. TH is a key enzyme involved in the biosynthesis of catecholamines, including dopamine, norepinephrine, and epinephrine. This antibody can be used to detect and quantify TH expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Mouse anti tubβiii, by Fortrea (2 mentions)
Alexa fluor 488 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse alexa fluor 594, by Abcam (1 mentions)
Mouse anti ctbp2, by BD (1 mentions)
Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Axiobserver z1, by Zeiss (1 mentions)
Rat anti cd11b, by Bio-Rad (1 mentions)
Mouse anti ctbp1, by BD (1 mentions)
Rabbit anti neun, by Cell Signaling Technology (1 mentions)
Fluoroshield mounting medium, by Abcam (1 mentions)
Rabbit anti gfap, by Merck Group (1 mentions)
Mouse anti β 3 tubulin, by Merck Group (1 mentions)
Mouse anti gfp, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
5 protocols using rabbit anti th
1
Immunostaining of Brain Slices and Cells
2
Immunofluorescence Staining of Neurospheres
3
Dopaminergic Neuronal Marker Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining for the dopaminergic neuronal markers class 3 beta tubulin (TUBβIII) and tyrosine hydroxylase (TH), the penultimate enzyme in the biosynthesis of dopamine20 (link),49 (link),50 (link), was performed on representative chips at day 24 of differentiation. Differentiated cells were fixed with 4% paraformaldehyde (PFA) in 1 × phosphate-buffered saline (PBS) for 15 min, by manually adding 70 μL in medium well inlet and 30 μL in medium well outlet followed by permeabilisation with 0.05% Triton-X 100 in 1 × PBS (3 min on ice), and blocking with 10% fetal calf serum (FCS) in 1 × PBS (1 h). After washing with 1 × PBS, the primary antibodies mouse anti-TUBβIII (1:2000, Covance) and rabbit anti-TH (1:2000, Santa cruz biotechnology), were incubated for 90 min at room temperature. After washing with 1 × PBS, the secondary antibodies Alexa Fluor 488 Goat Anti-Mouse and Alexa Fluor 568 Goat Anti-Rabbit together with a stain DNA (Hoechst 33342, Invitrogen), were incubated for 2 hours at room temperature. After washing with 1 × PBS and water, confocal images of representative culture chambers were acquired using a confocal microscope (Zeiss LSM 710).
4
Protein Quantification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of each sample was extracted using the cell lysis buffer for Western and IP (Beyotime, China). Forty μg protein was used for western blot analysis according to the following steps: Proteins were run on an SDS-PAGE (10% gel), then transferred to polyvinylidene fluoride (PVDF) membranes. They were incubated with rabbit anti-TH (Santa Cruz, CA, USA) followed by horseradish peroxidase-conjugated secondary antibodies. We determined the signals of bands using enhanced chemiluminescence (ECL kit, Amersham Biosciences, Piscataway, NJ, USA) and the Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, USA). The densitometric analysis was conducted with Image J 1.43 (National Institutes of Health). To ensure equal loading, blots were incubated with mouse anti-β-actin (Millipore, Billerica, MA, USA).
5
Characterizing Dopaminergic Phenotypes In Vitro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro dopaminergic phenotype is characterised by the expression of specific neuronal markers, TUBβIII and tyrosine hydroxylase (TH), the rate limiting enzyme in the biosynthesis of dopamine. 13, 34, 35 Immunostaining for TH positive cells was performed on representative wells at day 30 of differentiation. Differentiated cells were fixed with 4% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS) for 15 min, followed by permeabilisation with 0.05% Triton-X 100 in 1× PBS (3 min on ice), and blocking with 10% fetal calf serum (FCS) in 1× PBS (1 h). After washing with 1× PBS, the primary antibodies mouse anti-TUBβIII (1 : 2000, Covance) and rabbit anti-TH (1 : 2000, Santa Cruz Biotechnology) were incubated for 90 min at room temperature. After washing with 1× PBS, the secondary antibodies Alexa Fluor 488 Goat Anti-Rabbit and Alexa Fluor 568 Goat Anti-Mouse together with a stain DNA (Hoechst 33342, Invitrogen) were incubated overnight at 4 °C on a rotary shaker. After washing with 1× PBS, confocal images of representative culture chambers were acquired using a confocal microscope (Zeiss LSM 710). After the confocal images were acquired, a spot detection algorithm (Imaris software, Bitplane) was used to detect and count the nuclei in order to be able to calculate the efficiency of differentiation (see the ESI † for more details).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!