Senescence-associated β-Galactosidase (SABG) staining was performed following well-described methods53 (link). Basically, cells or tissues were fixed at room temperature in 2% formaldehyde/0.2% glutaraldehyde, washed, and incubated overnight at 37 °C with fresh SABG staining solution: 1 mg of 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-Gal) per mL (Fisher Scientific), 40 mM citric acid/sodium phosphate pH 6.0, 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 150 mM NaCl, and 2 mM MgCl2. Cells were washed and visualized and photographed under the microscope. Tissues were embedded in paraffin, sectioned and counterstained with Nuclear Fast Red or further used for immunohistochemistry.
5 bromo 4 chloro 3 indolyl beta d galactoside x gal
Manufactured by Thermo Fisher Scientific
5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-Gal) is a chromogenic substrate used for the detection of beta-galactosidase activity in various biological applications. It is a colorless compound that turns blue when cleaved by the beta-galactosidase enzyme, providing a visual indicator of enzymatic activity.
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3 protocols using 5 bromo 4 chloro 3 indolyl beta d galactoside x gal
1
Senescence-Associated β-Galactosidase Staining Protocol
2
Whole-mount X-gal Staining for SA-β-gal Activity
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Determination of SA–β–gal activity was performed through the widely used X-gal staining method. Whole-mount stainings were performed essentially as originally described [8 (link)]. Briefly, larvae between 2 and 11 dpf (n=10 for each developmental time) were fixed for 20 min (room temperature) in 2% formaldehyde/0.2% glutaraldehyde, washed and incubated overnight at 37ºC with freshly made SA– β–gal solution: 1 mg of 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-Gal) per mL (Fisher Scientific), 40 mM citric acid/sodium phosphate pH 6.0, 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 150 mM NaCl, and 2 mM MgCl2. After staining, some larvae were washed with PBS, mounted with glycerol and photographed with an Olympus microscope. Other animals were rinsed in PBS, cryoprotected with 30% sucrose in PBS overnight at 4 ºC, embedded in Tissue Tek (Sakura, Torrance, CA, USA), frozen in liquid nitrogen-cooled isopentane and cut serially on a cryostat (16 μm sections) in the transverse plane. Sections were mounted on Superfrost ® Plus glass slides (Menzel, Braunschweig, Germany) using Mowiol ® (Sigma).
3
Whole Mount SAbG Staining of Pkd2 Embryos
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Whole mount Senescence-Associated beta-Galactosidase (SAbG) staining was performed on E13.5 or E14.5 embryos derived from crosses of Pkd2 heterozygotic animals (a total of 4 WT and 3 null embryos). After extracting the uterine horns we separated each embryo from its placenta and embryonic sac and fixed them for 25 min (room temperature) in 2% formaldehyde/0.2% glutaraldehyde. After fixation, embryos were washed and incubated overnight at 37°C with fresh SAbG staining solution: 1 mg of 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-Gal) per mL (Fisher Scientific), 40 mM citric acid/sodium phosphate pH 5.5, 5 mM K 3 Fe[CN] 6 , 5 mM K 4 Fe[CN] 6 , 150 mM NaCl, and 2 mM MgCl 2 (Dimri et al., 1995) . Stained embryos were photographed on a magnifying glass, dehydrated in ethanol and embedded in paraffin. Blocks were serially sectioned at 5mm until the first blue stained section was observed. H&E staining was performed to confirm the presence of the desired organs (i.e. mesonephros and endolymphatic sac) and consecutive sections were obtained until blue staining disappeared for analysis.
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