Microdissected brain tissue samples were collected into 60 μl HEPES (Cat. No. 90909C, Sigma Aldrich) buffer containing 0.25% Protease-Inhibitor Cocktail Set III (Cat. No. 539134, Millipore, Billerica, MA, USA), and homogenized. Total protein content was determined in 5 μl of each sample, using a commercially available, colorimetric protein assay (Micro BCA Protein Assay Kit, Cat. No. 1856210, Thermo Scientific, Rockford, IL, USA), while approximately 30 μg protein per sample were analyzed in the western blot assay for TPH2 (56 kDa), using a 1:200 diluted antibody against TPH2 (rabbit anti-rat TPH2, Cat. No. ABN60, Millipore) and a horseradish peroxidase-conjugated secondary antibody (1:500, Cat. No. AP182P, Millipore), both in 5% dry milk TBST buffer. β-actin (42 kDa) served as the loading control, and blotted bands were visualized using chemoluminescence. See supplemental information for details.
Abn60
Manufactured by Merck Group
Sourced in United States
The ABN60 is a laboratory equipment product designed for general use in research and scientific settings. It serves as a versatile tool for various applications. The core function of the ABN60 is to provide a reliable and consistent platform for conducting experiments and analyses. No further details or interpretation of its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using abn60
1
Brain Tissue Protein Quantification and TPH2 Analysis
2
Immunohistochemistry of 5-HT, DsRed, and Tph2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunohistochemistry was performed as described previously (Sun et al., 2011) . Primary antibodies were anti-5-HT (1:500; Immunostar; 20079), anti-DsRed (1:500; Clontech; 632496), and anti-Tph2 (1:500; Millipore; ABN 60). Detailed experimental procedures are in Supplemental Experimental Procedures.
3
Immunohistochemical Staining of TH and TPH2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A polyclonal antibody to TH (catalog No. AB152, EMD Millipore Corporation, Billerica, MA, USA) and TPH2 (catalog No. ABN60, EMD Millipore Corporation) were used for immunohistochemistry as the primary antibody. Fluorescein isothiocyanate-labeled goat anti-rabbit IgG (catalog No. 65-6111, Invitrogen) was used as the secondary antibody. The immunohistochemical staining procedure was performed according to previously reported methods and conditions (Sutoo et al. 2002 (link)). Slices were rinsed with PBS four times for a total of 60 min. Primary antibodies were diluted 1:200 for TH and 1:400 for TPH2 with PBS and placed on each brain slice, then reacted for 12 h at 4 °C in a moist dark box. After reaction, the slices were rinsed four times with PBS for a total of 60 min. The secondary antibody was diluted 1:100 with PBS, placed on each slice, and incubated at 24 °C for 3 h in a moist dark box. After the reaction, the slices were rinsed four times with PBS for a total of 60 min. Finally, these slides were embedded in 10% glycerin in PBS and kept moist at 4 °C in a dark box.
4
Tissue Preparation and Staining for Brain Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse brain tissue was prepared by fixation via transcardial perfusion with 4% paraformaldehyde. Brains were then removed and equilibrated in graded sucrose solutions, frozen at −80°C in OCT solution, and cryosectioned at 25 μm for fluorescence/immunofluorescence imaging. Tissue processed in this way was suitable for imaging of endogenous protein fluorescence, immunofluorescence, and fluorescence in situ hybridization (FISH). Rat brains used in FISH experiments were harvested from unperfused animals, frozen at −80°C in OCT solution, and cryosectioned at 25 μm. FISH protocols for perfused mouse brains and unperfused rat brains both included paraformaldehyde fixation of sectioned tissue on slides. Primary antisera used included: rabbit anti-parvalbumin (PV-28, Swant, RRID:AB_2315235 ), rabbit anti-somatostatin (SST; T-4103, Peninsula, RRID:AB_518614 ), goat anti-choline acetyltransferase (ChAT; AB144P, EMD Millipore, RRID:AB_2079751 ), and rabbit anti-tryptophan hydroxylase (ABN60, EMD Millipore, RRID:AB_10806898 ). The endogenous fluorescence of GFP and tdTomato reporters was enhanced with goat anti-GFP (ab6673, Abcam, RRID:AB_305643 ); rabbit anti-GFP (A11122, Thermo Fisher, RRID:AB_221569 ); rabbit anti-red fluorescent protein (600-401-379, Rockland Immunochemicals, RRID:AB_2209751 ).
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!