What is LacZ staining?
What is LacZ staining?
LacZ is a frequently used reporter gene, encoding for the protein beta-galactosidase in cultured cells, which appear blue when the cultured cells are grown on a medium containing X-gal analog. The staining solution is made of X-gal (200 mg/ml), MgCl2 (1M), K ferri-cyanide (50mM), K ferro-cyanide (50mM), and PBS.
How do you stain paraffin sections?
Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature.
How do you detect LacZ?
The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-β-D: -galactoside (X-gal).
What is beta galactosidase staining?
The β-galactosidase Staining Kit provides an easy-to-use and efficient method to determine the transfection efficiency and expression of LacZ gene. β-galactosidase catalyzes the hydrolysis of X-gal, which produces a blue color in cells expressing the transfected gene.
Can you use the formalin-fixed paraffin-embedded sample in IHC staining?
An Overview of IHC Staining Procedures for Formalin-Fixed, Paraffin-Embedded (FFPE) Tissues. Over-fixation can render the tissues unusable for IHC. Ideally, tissues for IHC should be fixed for 24-48 hours, and then transferred to 70% ethanol until processing, embedding and sectioning.
How does lacZ reporter gene work?
The lacZ gene encodes beta-galactosidase, which catalyzes the cleavage of lactose to form galactose and glucose. Beta-galactosidase cleaves X-gal, a chromogenic substrate, resulting in an insoluble blue dye, thus allowing for the identification of cells with lacZ activity.
Where is beta-galactosidase found?
lysosomes
The GLB1 gene provides instructions for producing an enzyme called beta-galactosidase (β-galactosidase). This enzyme is located in lysosomes, which are compartments within cells that break down and recycle different types of molecules.
How does B gal staining work?
Why is beta-galactosidase important?
β-galactosidase is important for organisms as it is a key provider in the production of energy and a source of carbons through the break down of lactose to galactose and glucose. It is also important for the lactose intolerant community as it is responsible for making lactose-free milk and other dairy products.
What is the primary function of β galactosidase?
β-Galactosidase is an intracellular enzyme that is an essential part of the cellular metabolism of galactosides like lactose. It cleaves (separates) large substrate molecules into smaller ones by breaking the glycosidic bond. This enzyme is essential for energy production in most forms of multicellular life.
What kind of staining is used for LacZ?
The cleavage of Xgal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) results in a dark blue precipitate. A nuclear localized lacZ transgene can be used to mark transgene expressing cells unambiguously (endogenous enzyme activity is cytosolic). If desired, antibody staining can be carried out for cytosolic proteins (see Brinkmeier et al. ).
Can a lacZ transgene be used to mark cells?
A nuclear localized lacZ transgene can be used to mark transgene expressing cells unambiguously (endogenous enzyme activity is cytosolic). If desired, antibody staining can be carried out for cytosolic proteins (see Brinkmeier et al. ). Thick specimens, such as late stage mouse embryos, can be cleared by treatment with (see Turkay et al.).
How is beta galactosidase ( lacZ ) staining performed?
beta galactosidase (lacZ) Staining. This procedure describes how to process samples for lacZ staining. Beta galactosidase is an enzyme that hydrolizes beta galactosides. The cleavage of Xgal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) results in a dark blue precipitate.