Isolated mouse eyes were fixed in 3.7% formaldehyde overnight at 4 °C, cryoprotected in 30% sucrose solution for a minimum of 24 h prior to freezing and 20-μm thick cryosections cut. Sections were incubated in 0.25% Triton X-100 in DPBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 137.9 mM NaCl, 8.1 mM Na2HPO4–7 H2O [Corning, 21-0310CV]) for 10 min, followed by blocking buffer (5% goat serum, 0.5 g BSA in 50 ml DPBS) for 1 h prior to labeling. Samples were incubated sequentially in primary antibody at 4 °C overnight, followed by fluorescent-conjugated secondary antibody for 1 h at 37°C (Jackson ImmunoResearch Laboratories, 111-295-144, 115-545-003, 115-295-008). Primary antibodies used included: N-cadherin (BD Bioscience, 610921), β-catenin (BD Bioscience, 610154), E-cadherin (Cell Signaling, 24E10), βB-crystallin (Santa Cruz, FL-252), Rac1 (Abcam, ab155938), myosin-II (LifeSpan BioSciences, LS-C84042), WGA (LifeSpan BioSciences, LS-C76576), ZO-1 (Abcam, 61357), EphA2 (R&D, AF639), connexin 50 (ADI, Cx50-A), aquaporin-0 (ADI, AQP01-A), active Rac1 (NewEast Biosciences, 26903), p-myosin (Cell Signaling, 3674), and MLCK (Abcam, ab76092). F-actin was localized with Alexa448-conjugated phalloidin (Invitrogen-Molecular Probes). Nuclei were labeled with TO-PRO-3 (Invitrogen-Molecular Probes).
Alexa448 conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Alexa448-conjugated phalloidin is a fluorescent conjugate used for the detection and visualization of actin filaments in cells. It binds specifically to f-actin, allowing for the labeling and imaging of the actin cytoskeleton.
Automatically generated - may contain errors
2 protocols using alexa448 conjugated phalloidin
1
Immunohistochemical Analysis of Mouse Eye Tissue
2
Immunohistochemical Analysis of Lens Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated lenses, lenses from organ culture, and primary lens cell cultures were fixed in 3.7% formaldehyde (15 min for primary cultures and overnight for whole lenses). For detecting phospho-protein, cultures were pre-treated with pervanadate for 20 min prior to fixation. Whole lenses were cryopreserved in 30% sucrose solution after fixation for 24 h or longer and prepared for cryosectioning. 20-μm thick sections were cut. Samples were then permeabilized with 0.25% Triton X-100 in DPBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 137.9 mM NaCl, 8.1 mM Na2HPO4–7 H2O [Corning, 21-0310CV]) for 10 min, blocked in blocking buffer for 1 h (5% goat serum, 0.5 g BSA in 50 ml DPBS) and then incubated sequentially in primary antibody either for 3 h at 37 °C or overnight at 4 °C, followed by a fluorescent-conjugated secondary antibody for 2 h (Jackson ImmunoResearch Laboratories, 111-295-144, 115-545-003, 115-295-008). F-actin was localized with Alexa448-conjugated phalloidin (Invitrogen-Molecular Probes, A12379). Nuclei were counterstained with TO-PRO-3 (Invitrogen-Molecular Probes, T3605). All sections were cut serially in the anterior to posterior direction.
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!