Mice were perfused transcardially with PBS followed by 4% PFA in PBS. For whole-mount retinal staining, mice were injected intravitreally with 1 µL MitoTracker Deep Red (500 nM) 20 minutes before the perfusion. Eyeballs were enucleated and fixed in 4% PFA for 15 minutes. The retina was dissected out, put in 30% sucrose overnight at 4°C, and used for staining. The following antibodies were used: anti-HA (1:500; Abcam, Cambridge, MA), anti-RBPMS (1:200; PhosphoSolutions, Aurora, CO), anti-active CASPASE3 (1:200; Abcam), anti-rabbit IgG 546 (1:600; Invitrogen, Carlsbad, CA), anti-mouse IgG 488 (1:600; Invitrogen), and anti–guinea pig 647 (1:600; Invitrogen). Imagines were taken with a Leica TCS SP5 confocal microscope.
Anti mouse igg 488
Manufactured by Thermo Fisher Scientific
Anti-mouse IgG 488 is a secondary antibody labeled with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize mouse primary antibodies in various applications, such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg 488, by Thermo Fisher Scientific (2 mentions)
Anti ha, by Abcam (1 mentions)
Anti active caspase 3, by Abcam (1 mentions)
Anti rbpms, by PhosphoSolutions (1 mentions)
Tcs sp5 confocal microscope, by Leica camera (1 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
Dapi counterstain, by Southern Biotech (1 mentions)
Donkey anti goat 488, by Thermo Fisher Scientific (1 mentions)
Goat anti cd31, by R&D Systems (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Stereo investigator software, by MicroBrightField (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Lsm 700 exciter, by Zeiss (1 mentions)
Paraformaldehyde solution, by Biosesang (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using anti mouse igg 488
1
Retinal Mitochondrial Imaging in Mice
2
Quantifying Infarct Volume and Microvasculature
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Perfused fixed brains were cryopreserved in 25% sucrose and then embedded in OCT and serial cryosectioned. Infarct volume (mm3) was assessed by the Cavalieri Estimator probe using the StereoInvestigator software (MicroBrightField, Williston, VT, USA), as previously described. Briefly, six serial coronal sections, cut at 30 μm, were stained using a 0.2% Cresyl violet solution (Electron Microscopy Science, Hatfield, PA) or anti-mouse IgG-488 (Invitrogen, Waltham, MA). The total volume of infarct was quantified by estimating the area of tissue loss in the ipsilateral cortical hemisphere using six, serial coronal sections. A 100 μm spaced grid was placed over the ipsilateral hemisphere in the Cavalieri probe and infarcted area scored. The coronal sections were used for immunohistochemical (IHC) staining of CD31. Sections were blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO) in 0.2% Triton-X100, incubated with goat anti-CD31 (1 : 100, R&D systems, Minneapolis, MN, USA) overnight at room temperature (RT), washed with 1X PBS, and then incubated with donkey anti-goat 488 (1 : 250, Thermofisher, USA) for 1 hour at RT. Slides were then mounted with DAPI counterstain (SouthernBiotech, Birmingham, AL), and images were acquired using Olympus fluorescence microscope. Microvessel diameters were measured using ImageJ.
3
Immunofluorescent Staining of HIF-1α and pCREB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 25 μl of 4% paraformaldehyde per well (10 min), permeabilized for 10 min (PBS/0.5% triton-X 100), and washed once with PBS/0.05% Tween-20. Then, cells were incubated for 1 h with primary antibodies (anti-HIF-1α, 1:100, BD Bioscience #610958, anti-phospho CREB, 1:200, CST #9198S) diluted in blocking buffer (PBS/0.05% Tween 20/5% bovine serum albumin). After three washes in wash buffer (PBS/0.5% Tween-20), cells were incubated for 1 h with secondary antibodies (anti-Mouse IgG 488, 1:1000 Invitrogen, #A11029; anti-rabbit IgG 488, 1:1000, Invitrogen, #A11034) followed by DAPI staining (5 μg/ml).
4
Immunofluorescence Imaging of AhR and Annexin V
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After washing with PBS containing 0.1% BSA (Sigma-Aldrich), cells were fixed in 4% paraformaldehyde solution (Biosesang, Seongnam, Korea) at 4°C. After 20 min, cells were washed with PBS containing 0.1% BSA followed by permeabilization solution containing PBS with 0.1% BSA and 0.3% Triton X-100 (Sigma-Aldrich). After 10 min, antibodies were added at the following dilutions: human AhR (Abcam, Cambridge, MA, USA) 1 : 200 and Annexin V (Millipore, Burlington, USA) 1 : 200. Next, PBS containing 0.1% BSA and 10% FBS was dispensed to the cells. The cells were incubated overnight at 4°C. Then, fluorescence-labeled secondary antibodies were used as follows: anti-rabbit IgG 594 (1 : 400) (Invitrogen) and anti-mouse IgG 488 (1 : 400) (Invitrogen). After washing, cells were costained with 4′6-diamidino-2-phenylindole (DAPI) at 1 : 1000 (Sigma-Aldrich) for 6 min. Fluorescence images were taken using a Confocal Laser Scanning Microscope (Carl-Zeiss LSM 700 Exciter, Oberke, Germany). One representative of 3 independent experiments is shown.
5
Quantifying HIF-1α and pCREB Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 25μl of 4% paraformaldehyde per well (10 min), permeabilized for 10 min (PBS/0.5% triton-X 100), and washed once with PBS/0.05% Tween-20. Then, cells were incubated for 1 h with primary antibodies (anti-HIF-1α, 1:100, BD Bioscience #610958, anti-phospho CREB, 1:200, CST #9198S) diluted in blocking buffer (PBS/0.05% Tween 20/ 5% bovine serum albumin). After three washes in wash buffer (PBS/0.5% Tween-20), cells were incubated for 1 h with secondary antibodies (anti-Mouse IgG 488, 1:1000 Invitrogen, #A11029; anti-rabbit IgG 488, 1:1000, Invitrogen, #A11034), followed by DAPI staining (5 μg/ml). or relative (B) distance was analyzed. Expression profiles of genes whose distance changed significantly in the radial distance analysis is shown as a fold induction (log2) upon hypoxic treatment (A, left). Green: up-regulated genes, red: down-regulated genes. Expression level of the two genes in the pair was expressed as the product and sum of the induction level of two genes in the relative distance analysis (B, left).
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!