Cells were harvested in PBS containing 2 mM N-ethylmaleimide (NEM; Sigma-Aldrich, E3876) and lysed in Tris-buffered saline (Sigma-Aldrich, T6664) containing 1% SDS (Sigma-Aldrich, L3771) and 20 mM NEM 30 h after transfection. The lysate was boiled, sonicated, and centrifuged at 14,000 × g for 15 min. The supernatant was diluted in NP-40 buffer containing 2 mM NEM, and immunoprecipitation was carried out using standard methods.
T6664
Manufactured by Merck Group
Sourced in United Kingdom
T6664 is a laboratory equipment product manufactured by Merck Group. It is a general-purpose device designed for use in scientific research and analytical applications. The core function of T6664 is to perform specific tasks or procedures within a controlled laboratory environment.
Automatically generated - may contain errors
3 protocols using t6664
1
Protein Extraction and Immunoprecipitation
2
Immunohistochemical Analysis of Dmrt1, Erβ, and Sox9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Dmrt1, estrogen receptor β (Erβ), and Sox9 proteins, immunohistochemical staining was performed following the methodology described by Kamaszewski et al. [17 (link),24 (link)]. The histological preparations obtained were deparaffinized in xylene and then rehydrated in a gradient of ethanol. The activity of endogenous peroxidase was blocked by 10 min incubation of the specimens with a 3% solution of hydrogen peroxide. The histological slides were then rinsed with Tris buffer (pH 8.0) (T-6664, Sigma). Incubation with primary antibodies was carried out in a humid chamber according to the parameters shown in Table 2 .
The visualization was performed using the EnVision + System–HRP (DAKO, Glostrup, Denmark) according to the manufacturer’s instruction. Cell nuclei were stained with the Harris hematoxylin. During the immunohistochemical reaction, the negative control, not incubated with the primary antibody, was used. Subsequently, the specimens were dehydrated in a gradient of ethanol, cleared with xylene, and mounted with DPX. (Sigma-Aldrich, St. Louis, MO, USA).
The visualization was performed using the EnVision + System–HRP (DAKO, Glostrup, Denmark) according to the manufacturer’s instruction. Cell nuclei were stained with the Harris hematoxylin. During the immunohistochemical reaction, the negative control, not incubated with the primary antibody, was used. Subsequently, the specimens were dehydrated in a gradient of ethanol, cleared with xylene, and mounted with DPX. (Sigma-Aldrich, St. Louis, MO, USA).
3
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with Tris-Buffered Saline (TBS) (TBS; T6664; Sigma-Aldrich, Dorset, UK) for 2 minutes prior to fixation in 4% formaldehyde (28908, Thermo Scientific, Massachusetts, USA) at 4C for 1 hour. Cells were then washed with TBS and permeabilized with 0.1% Triton X 100 (85111, ThermoFisher, Massachusetts, USA) for 30 minutes. Following a further TBS wash, samples were blocked with 5% goat serum (G6767; Sigma-Aldrich, Dorset, UK) for 1 hour. Primary antibody was then added and incubated at 4C overnight (Primary antibodies listed in Table 1) after which, cells were washed in TBS on a shaker for approximately 20 minutes and then washed again before adding secondary antibody, Hoescht and phalloidin.
Secondary antibody incubation was at 4C for 4 to 24 hours, depending on antibody, and three further wash steps were performed after incubation and before microscopy.
Secondary antibody incubation was at 4C for 4 to 24 hours, depending on antibody, and three further wash steps were performed after incubation and before microscopy.
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!