Mice were deeply anesthetized and transcardially perfused with a saline solution followed by 4%paraformaldehyde in 0.1-M phosphate buffer [57 (link)]. Staining was performed by incubating slides with primary antibody (rabbit anti-TH, 1:500, abcam, UK) over night, followed by incubation with secondary antibody (Alexa fluor 488, Invitrogen 1:1000). The total number of TH-expressing neurons in each LC was estimated taking into account the cell group contained in 7 * 10 μm2. For each animal, the number of TH-positive neurons were counted from the right and left LC and the mean between both sides was done for each mouse. Pictures were performed using Leica microscope.
Rabbit anti th
Manufactured by Abcam
Sourced in United States
Rabbit anti-TH is a primary antibody that specifically binds to the tyrosine hydroxylase (TH) protein. TH is an enzyme involved in the biosynthesis of catecholamine neurotransmitters, including dopamine, norepinephrine, and epinephrine. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and study the expression and localization of TH in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Freezing microtome, by Leica (1 mentions)
Plan apo, by Nikon (1 mentions)
Phentolamine, by Merck Group (1 mentions)
Guanethidine, by Merck Group (1 mentions)
Tetrodotoxin, by Merck Group (1 mentions)
Chicken anti th, by Abcam (1 mentions)
Acetylcholine, by Merck Group (1 mentions)
Phenylephrine, by Merck Group (1 mentions)
Sodium nitroprusside, by Merck Group (1 mentions)
Rabbit anti mao b, by Abcam (1 mentions)
Mouse anti trk b, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
Rabbit anti iba1, by Abcam (1 mentions)
Anti rabbit igg alexa flour 488, by Abcam (1 mentions)
Rabbit anti gfap, by Proteintech (1 mentions)
D9663, by Merck Group (1 mentions)
Fluoromount g mounting medium, by Southern Biotech (1 mentions)
Nuclear dye hoechst 33342, by Merck Group (1 mentions)
9 protocols using rabbit anti th
1
TH-Positive Neurons Quantification in Locus Coeruleus
2
Immunohistochemical Analysis of Substantia Nigra
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thirty-two days postsurgery, rats were sacrificed and transcardially perfused with a 4% paraformaldehyde solution (PFA). After overnight postfixation in the same fixative, the brains were transferred to 30% sucrose in PBS for cryoprotection. Then, the brains were snap-frozen and cut at a 40 μm section thickness on a Leica freezing microtome. The substantia nigra pars compacta (SNpc) sections and the striatum sections were cut according to the rat brain Atlas by George Paxinos and Charles Watson (sixth edition). Sections were collected and stored at 4°C.
For immunohistochemistry, sections were moved to 5% H2O2 in PBS for 15 min to quench endogenous peroxidase activity. After washing, sections were sequentially treated with 0.25% Triton-X 100 in PBS, followed by blocking with 5% goat serum for 1 h and 30 min. They were then immunostained with specific antibodies overnight at 4°C, the primary antibodies being as follows: rabbit anti-TH (Abcam, 1 : 200). Sections were then incubated with fluorescent-labeling goat antirabbit secondary antibody (Boster, 1 : 200) for 2 hours in the dark at room temperature after washing in PBS twice. Finally, sections were viewed by fluorescence microscopy (Olympus BX51, Japan).
For immunohistochemistry, sections were moved to 5% H2O2 in PBS for 15 min to quench endogenous peroxidase activity. After washing, sections were sequentially treated with 0.25% Triton-X 100 in PBS, followed by blocking with 5% goat serum for 1 h and 30 min. They were then immunostained with specific antibodies overnight at 4°C, the primary antibodies being as follows: rabbit anti-TH (Abcam, 1 : 200). Sections were then incubated with fluorescent-labeling goat antirabbit secondary antibody (Boster, 1 : 200) for 2 hours in the dark at room temperature after washing in PBS twice. Finally, sections were viewed by fluorescence microscopy (Olympus BX51, Japan).
3
MPTP-Induced Neurodegeneration in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously reported,5 (link) adult male C57Bl/6 mice were individually housed
for 1
week and then injected IP daily for 5 days with 30 mg kg–1 day–1 free-base MPTP (Sigma). On day 6, 24 h after
the mice received the fifth and final dose of MPTP, daily treatment
with compound was initiated. Mice received twice-daily doses of each
compound (or vehicle) for the following 21 days, after which mice
were sacrificed by transcardial perfusion with 4% paraformaldehyde.
The brains were dissected, fixed overnight in 4% paraformaldehyde,
and cryoprotected in sucrose for freezing by standard procedures.
Frozen brains were sectioned through the striatum and SNc at 30 μM
intervals, and every fourth section (spaced 120 μM apart) was
stained with antibodies directed against tyrosine hydroxylase (TH)
(Abcam, rabbit anti-TH, 1:2500). Diaminobenzidine was used as a chromagen,
and tissue was counter-stained with hematoxylin to aid in visualization
of the neuroanatomy. Images were analyzed with a Nikon Eclipse 90i
motorized research microscope with Plan Apo lenses coupled with Metamorph
Image Acquisition software (Nikon). TH+ neurons were counted with
ImageJ software (NIH) in every section by two blinded investigators,
and the results were averaged and multiplied by the sectioning interval
to determine the total number of TH+ neurons per SNc.
for 1
week and then injected IP daily for 5 days with 30 mg kg–1 day–1 free-base MPTP (Sigma). On day 6, 24 h after
the mice received the fifth and final dose of MPTP, daily treatment
with compound was initiated. Mice received twice-daily doses of each
compound (or vehicle) for the following 21 days, after which mice
were sacrificed by transcardial perfusion with 4% paraformaldehyde.
The brains were dissected, fixed overnight in 4% paraformaldehyde,
and cryoprotected in sucrose for freezing by standard procedures.
Frozen brains were sectioned through the striatum and SNc at 30 μM
intervals, and every fourth section (spaced 120 μM apart) was
stained with antibodies directed against tyrosine hydroxylase (TH)
(Abcam, rabbit anti-TH, 1:2500). Diaminobenzidine was used as a chromagen,
and tissue was counter-stained with hematoxylin to aid in visualization
of the neuroanatomy. Images were analyzed with a Nikon Eclipse 90i
motorized research microscope with Plan Apo lenses coupled with Metamorph
Image Acquisition software (Nikon). TH+ neurons were counted with
ImageJ software (NIH) in every section by two blinded investigators,
and the results were averaged and multiplied by the sectioning interval
to determine the total number of TH+ neurons per SNc.
4
Striatal Protein Expression Analysis in Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The striatum of rats was harvested and washed with ice-cold NS solution, followed by lysis in a lysis buffer on ice for 30 min. Western blot analysis was finally performed on samples obtained from a total of 30 rats. After centrifugation at 12,000×g for 30 min, protein concentration was determined using the BCA protein assay. Lysate protein (20 μg) was separated by SDS-PAGE on a 12 % gel and transferred to an NC membrane. After blocking with 5 % nonfat milk for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C: rabbit anti-TH (1:200, Abcam, Cambridge, MA, USA) and mouse anti-β-actin (TA-09, Zhongshanjinqiao, Inc., Beijing, China). Following primary antibody incubation, the membranes were then incubated with the respective secondary antibodies, either goat anti-mouse IgG or goat anti-rabbit IgG (Zhongshanjinqiao), for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) and analyzed using Image J.
5
Neurochemical Reagents and Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetylcholine, guanethidine, phentolamine, phenylephrine, sodium nitroprusside and tetrodotoxin were purchased from Sigma Aldrich Chemicals Co. (Missouri, USA). Rabbit anti-S100p was obtained from Novocastra/Leica Biosystems (Newcastle, UK). Rabbit anti-TH, chicken anti-TH, goat anti-chicken gamma immunoglobulin (IgG) and a rabbit anti-goat IgG were purchased from Abcam (Cambridge, USA).
6
Neurochemical Markers in Brain and Gut
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After anesthesia, each animal was sacrificed, and the brain and intestine regions of interest were collected and then immediately frozen at −80°C for subsequent analysis (N = 10). The following primary antibodies were applied: (a) rabbit anti-ZO-1 (1:2000, Proteinetch Group, Chicago, IL, United States); (b) mouse anti-Trk B (1:2000, Santa Cruz Biotechnology, CA); (c) rabbit anti-TPH (1:500, Abcam, Cambridge, MA); (d) rabbit anti-TPH2 (1:1000, Abcam); (e) mouse anti-IDO1 (1:5000, Proteinetch); (f) rabbit anti-MAOA (1:5000, Abcam); (g) rabbit anti-MAOB (1:3000, Abcam); (h) rabbit anti-TH (1:500, Abcam); (i) rabbit anti-BDNF (1:4000, Abcam). The detailed extraction and detection methods are described in the Supplementary Materials.
7
Immunohistochemical Analysis of Neuronal Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were perfused intracardially with PBS and subsequently fixed with 4% paraformaldehyde. After tissue was dehydrated and embedded in paraffin, a total of 5-μm section was cut and collected. Antigens were repaired via high temperature and pressure antigen repair using citrate buffer (pH=6). Tissue was cooled to room temperature and incubated in goat serum for 30 min, and incubated with the following primary antibodies at 4 overnight: rabbit anti-TH (1:500; Abcam), rabbit anti-IBA-1 (1:300; Abcam), rabbit anti-GFAP (1:500; Proteintech), rabbit anti-ΔFOS B (1:100; HuaAn Biotechology). For immunohistochemical staining, slides were incubated with biotinylated secondary antibody at 37°C for 15 min and visualized with avidin horseradish enzyme at 37°C for 20 min. Slices were visualized through DAB kit. For immunofluorescence staining, the second antibodies were anti-rabbit IgG Alexa Flour 488 (1:800; Abcam). Quantification of TH-positive neurons was performed through visually counting the number of TH-positive neuronal cell bodies blindly by two investigators. The images were recorded with a charge-coupled device camera and operated with the MetaMorph software. The results were obtained from the average. The mean value for the number of TH-positive neurons in substantia nigra was then deduced by averaging the counts of six sections for each rat.
8
Immunostaining of Cultured hMOs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured and fixed hMOs were embedded in a 3% lowmelting-point agarose (Biozym) in PBS. Subsequently, 50 µm thick sections were cut using a vibratome (Leica VT1000s) and center-sections were used for assessing TH/FOXA2/TUJ1 expression. Prior to the immunostaining, sections were permeabilized using 0.5% Triton X-100 in PBS. Depending on the antibody, permeabilization times varied between 30 min and 2 h. Unspecific antigen blocking was achieved by incubating cut sections for 2 h in 2.5% donkey serum (Sigma-Aldrich, D9663), 2.5% BSA, 0.1% Triton X-100 and 0.1% sodium azide, followed by primary antibody incubation at 4 °C for 48 h on a shaker. Antibodies were diluted in blocking buffer as follows: rabbit anti-TH (1 : 1000, Abcam), chicken anti-TUJ1 (1 : 600, Millipore). This was followed by the incubation with secondary antibodies diluted in PBS containing 0.01% Triton X-100 and Hoechst-33342 nuclear dye (1 : 1000, Sigma-Aldrich). All secondary antibodies (Invitrogen) were conjugated to Alexa Fluor fluorochromes. Sections were mounted in Fluoromount-G mounting medium (Southern Biotech) and analyzed employing a confocal laser scanning microscope (Zeiss LSM 710).
9
Immunohistochemical Analysis of Rat Brain Regions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the end of the experiment, rat brains were subjected to an immunohistochemical analysis using antibodies for tyrosine hydroxylase as previously described 8 . Brain sections (40 µm) were incubated with normal goat serum followed by rabbit anti-TH (Abcam Inc., Cambridge, MA, USA), and then biotinylated goat anti-rabbit IgG secondary antiserum (Vector laboratories, Inc. Burlingame, CA, USA) was applied. Incubation was carried out with peroxidase-conjugated streptavidin (Vector laboratories, Inc. Burlingame, CA, USA). During incubations, sections were rinsed and reacted with 3,3'-diaminobenzidine (DAB) as a chromogen. After mounting sections on gelatin-chromalum-coated slides, sections were dehydrated and coverslipped. The sites for stimulation/recording with the tungsten electrodes were also confirmed in every animal's brain at the end of the experiments. Only data from rats in which the location of electrode tips was found in the ACC or the PAG region were used for the data analysis.
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!