E16.5 inner ears or eyes were fixed for 1 hour in PFA 4%, equilibrated in sucrose 20% overnight and embedded in a 1:1 mixture of sucrose 20%: OCT (Tissue-Tek). Cryosections (12μm) were stored at −80C, thawed, and processed for citrate antigen retrieval (boiled five times for 1 minute in 10mM citrate with cooling intervals). The sections were then permeabilized and blocked in 0.5% Triton X-100 and 1% BSA, and incubated with the primary antibodies overnight (NR2F1: R&D Systems PPH813299, mouse monoclonal clone H8132 (1:100); SOX2: Santa Cruz Biotechnology sc-17320, goat polyclonal (1:100)). Secondary antibodies were donkey anti-mouse conjugated to Alex Fluor 555 and donkey anti-goat conjugated to Alex Fluor 647 (Thermofisher). Nuclei were stained with Hoechst 33342 (Thermofisher). For the quantifications in Fig. 3D , a 25×25 μm region of interest was defined to match the organ of Corti, and signal intensity was measured separately for the SOX2 and NR2F1 channels using ImageJ (IntDens). For whole-mounts at birth (Fig. 5 ), inner ears were fixed 1 hour in 4% PFA, the sensory epithelia were exposed, permeabilized in 0.5% Triton X-100, stained with phalloidin conjugated to Alexa Fluor 488 (Thermofisher), and mounted flat. Images were acquired with a Zeiss LSM800 confocal microscope.
Nr2f1
Manufactured by R&D Systems
NR2F1 is a nuclear receptor that functions as a transcriptional regulator. It is involved in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Sc 17320, by Santa Cruz Biotechnology (1 mentions)
Alex fluor 647, by Thermo Fisher Scientific (1 mentions)
Alex fluor 555, by Thermo Fisher Scientific (1 mentions)
Nestin, by Abcam (1 mentions)
N cad, by Abcam (1 mentions)
Prdm1, by Cell Signaling Technology (1 mentions)
Nanog, by Abcam (1 mentions)
2 protocols using nr2f1
1
Immunohistochemistry of Inner Ear and Eye
2
Immunofluorescence Staining of Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4% paraformaldehyde in DPBS for 20 minutes and permeabilized with 1% Triton X-100 for 20 minutes at room temperature. After 60 minutes blocking with 2% normal goat serum, cells were incubated with primary antibody overnight at 4°C, washed, and stained with secondary antibodies (1:300, goat anti-rabbit IgG-Cy3; or 1:300, goat anti-mouse IgG-Cy3) for 60 minutes at room temperature and then washed three times with phosphate-buffered saline (PBS). The primary antibodies for respective cells include OCT4 (1:200, Abcam), NANOG (1:200, Abcam), PAX6 (1:200, Abcam), SOX2 (1:200, Abcam), NESTIN (1:200, Abcam), SOX1 (1: 200, Abcam), ZO-1 (1:100, Abcam), N-CAD (1: 100, Abcam), MAFB (1:300, Sigma), SOX9 (1:300, EMD Millipore), PRDM1 (1:200, Cell Signaling Technology), and NR2F1 (1:300, R&D Systems). DAPI (4', 6-diamidino-2-phenylindole) (1:500) was used as counter-staining for nuclei. The images were captured and analyzed with the Olympus IX73 and Image J.
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!