Samples were harvested, fixed in 0.2% glutaraldehyde solution overnight at 4°C, cryoprotected in 30% sucrose solution and embedded in OCT. Sections were dried at room temperature for 30 minutes, rehydrated in PBS for 5 minutes and post‐fixed in 0.2% glutaraldehyde solution for 15 minutes at room temperature. Slides were then washed 3 × 15 minutes in the washing buffer containing 1M MgCl2 (Sigma‐Aldrich; ref M8266), 1% Na‐deoxycholate (Sigma‐Aldrich; ref D6750), 2% NP40 (ref 74385, diluted in H2O) in PBS. Sections were incubated overnight at 37°C in a humidified chamber in X‐Gal solution containing X‐Gal (Thermo Fisher Scientific; ref R0404; 50 mg/mL in DMSO), 1X potassium ferrocyanide, 1X potassium ferrocyanide, 1M Tris (pH 7.3–7.4) diluted in washing buffer. Sections were washed in PBS 3 × 5 minutes, counterstained with 1% eosin for 2 minutes, dehydrated, and mounted with NeoMount® mounting medium.
X gal
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania, Canada
X-gal is a colorimetric substrate used in molecular biology and microbiology applications. It is commonly used to detect the presence of beta-galactosidase enzyme activity in cells or organisms.
Automatically generated - may contain errors
Lab products found in correlation
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (10 mentions)
Glutaraldehyde, by Merck Group (7 mentions)
Ckx41 inverted light microscope, by Olympus (6 mentions)
Mgcl2, by Merck Group (5 mentions)
Kanamycin, by Merck Group (5 mentions)
K3fe cn 6, by Merck Group (4 mentions)
Tetracycline, by Merck Group (4 mentions)
Na deoxycholate, by Merck Group (3 mentions)
Nuclear fast red, by Merck Group (3 mentions)
Nuclear fast red, by Vector Laboratories (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sodium hydroxide (naoh), by Merck Group (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Laemmli buffer, by Merck Group (3 mentions)
104 protocols using x gal
1
Tissue Preparation and X-Gal Staining
2
Senescence-Associated Beta-Galactosidase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in six-well plates until day 7, fixed with 0.5% (w/v) glutaraldehyde (Sigma) in PBS for 10–15 min, washed with 1 mM MgCl2/PBS (pH 6.0), and then incubated with X-Gal staining solution (1 mg/mL X-Gal, Thermo Scientific), 5 mM K3[Fe(CN)6], and 5 mM K4[Fe(CN)6] for 8 h at 37°C. Bright-field images of cells were taken using a DP20 digital camera attached to the Olympus CKX41 inverted light microscope. The percentage of SA-β-Gal-positive cells was estimated by counting at least 100 cells per replicate.
3
Brain LacZ Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gt(ROSA)26Sor-LacZ mice and Nes-cre; Gt(ROSA)26Sor-LacZ mice were transcardially perfused with phosphate buffered saline (PBS), followed by PFA 4% in PBS at RT. Brains were dissected and cryoprotected in 30% sucrose solution (in PBS) overnight at 4°C. Floating brain sections (50 µm) were collected in PBS and stained with X-Gal staining solution for 5 min at 30°C in the dark and washed in PBS. X-Gal staining solution in PBS: 2 mg/ml X-Gal (ThermoFisher Scientific), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.25% Triton X-100.
4
β-Galactosidase Activity in Rapamycin-Treated HCECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
X-gal staining for β-galactosidase activity was performed on HCEC treated with rapamycin 2 nM and control (DMSO) at week two and week five. Each sample was rinsed twice with PBS, fixed in 3.7% PFA for 15 minutes. Cells were washed with PBS, and then stained for 36 hours at 37 °C in X-gal solution with PH of 6.0 containing 1 mg/ml X-gal (Thermo Scientific), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2. X-gal positive cells (cells with greenish color) were examined in 10 fields from each sample under light microscope (Carl Zeiss, Thornwood, NY), and photographed with an AxioCam (Carl Zeiss) camera.
5
Quantifying Senescence-Associated β-Galactosidase
6
β-galactosidase Staining of Myotubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staining for β-galactosidase was performed using modifications of a protocol previously published by others (47 (link)). In brief, after myotubes grown on a coverslip were washed, they were washed in PBS, were fixed for 5 minutes with 3% formaldehyde, were washed and incubated at 37°C with fresh X-Gal (Thermo Fisher Scientific) working solution (1:40 dilution in X-Gal dilution buffer: potassium ferricyanide crystalline 5 mM, potassium ferricyanide trihydrate 5 mM, magnesium chloride 2 mM in PBS protected from light) for 12 hours, and were rinsed with PBS for 5 minutes and then briefly with distilled water. Nuclear fast red was used for counterstaining for 5 minutes, and myotubes were mounted with aqueous mounting medium.
7
X-gal Staining of Embryonic Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The embryos or small tissue pieces were dissected in PBS on ice and then fixed with 4% paraformaldehyde overnight at 4°C. Samples were rinsed with PBS 3 times and then incubated at 37°C for 2 to 4 hours in X-gal (Thermo Fisher Scientific, B1690) solution diluted in the X-gal Reaction Buffer (35 mM potassium ferricyanide, 2 mM MgCl2, 0.02% Nonidet P-40, 0.01% Na deoxycholate). After the incubation, the samples were rinsed with PBS until the solution was no longer yellow.
8
X-gal Staining and H&E Tissue Sections
9
Senescence-Associated β-Galactosidase Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Detection of senescence associated β-galactosidase (SA-β-Gal) activity was performed as described [42 (link)]. Immediately before staining, X-Gal stock solution was prepared by dissolving 20mg/ml X-Gal (Invitrogen, Carlsbed, CA) in dimetylformamide. SA-β-Gal staining solution was prepared as follows: 1 mg/ml of X-Gal stock solution were dissolved in 40 mM citric acid in sodium phosphate, pH 6.0/5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl2. Frozen sections of 10-μm thickness were fixed for 5′ in 4% formaldehyde/0.5% glutaraldehyde at 4°C, washed in PBS and incubated in fresh SA-β-Gal staining solution for 16h at 37°C. No special blocking step is required to perform the staining. Sections were counterstained with Hematoxylin.
10
Senescence-Associated β-Galactosidase Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staining for SA-β-gal was performed according to published procedures [50 (link)]. Briefly, X-Gal stock solution was prepared by dissolving 20mg/ml X-Gal (Invitrogen, Carlsbed, CA) in dimetylformamide, immediately before staining. SA-β-Gal staining solution was prepared as follows. One mg/ml of X-Gal stock solution were dissolved in 40 mM citric acid in sodium phosphate, pH 6.0/5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl2. Frozen sections of 10-μm thickness were fixed for 5′ in 4% formaldehyde/0.5% glutaraldehyde at 4°C, washed in PBS and incubated in fresh SA-β-Gal staining solution for 16h at 37°C. Sections were counterstained with Hematoxylin.
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!