For retinal flat-mount staining, eyes from P12 and P17 mice were fixed in 4% PFA for 1 hour, and retinas were then dissected out. After permeabilization with 0.5% Triton X-100/PBS for 1 hour at RT, retinas were incubated with Isolectin GS-IB4 (20 µg/mL; Invitrogen, Carlsbad, CA, USA) for 2 hours at RT. After being washed in PBS, retinas were flat-mounted with 60% glycerol in PBS. Images were taken with a laser scanning confocal microscope (DMI6000B with TCS SP8 system; Leica, Wetzlar, Germany) at 10× magnification. The relative neovascular area and avascular area were analyzed with ImageJ software. The Freehand tool was used to select the edge of the retina and vaso-obliteration area. For the neovascular area, captures were inverted and shifted to 8-bit, and the threshold was set at 225. The ratios calculated were vaso-obliteration area to retina area and neovascular area to retina area.
Dmi6000b with tcs sp8 system
Manufactured by Leica
Sourced in Germany
The DMI6000B with TCS SP8 system is a high-performance microscope platform designed for advanced imaging and analysis. It combines a robust inverted microscope (DMI6000B) with the highly versatile TCS SP8 confocal laser scanning system. The core function of this equipment is to provide researchers with a powerful tool for conducting detailed and precise observations and measurements of microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
Isolectin gs ib4, by Thermo Fisher Scientific (1 mentions)
Mab1398z, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Ab211542, by Abcam (1 mentions)
2 protocols using dmi6000b with tcs sp8 system
1
Retinal Flat-Mount Staining Protocol
2
Assessing p16INK4a Expression in Mouse Eyes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eyes were fixed in 4% paraformaldehyde for 1 h at RT and then dehydrated in 30% sucrose solution overnight. Afterward, dehydrated eyes were embedded in Tissue-Tek® optimum cutting temperature compound (Sakura Finetek, Japan) and cross-sectioned on a cryostat vertically through the center of the cornea and optic nerve. Serial 15-μm-thick frozen eyes sections were cut using a cryostat (NX70, Thermo Fisher Scientific, US). The eye sections were permeabilized with 0.3% Triton X-100/PBS for 30 min at RT, blocked with 2% BSA, and 0.3% Triton X-100 PBS for 1 h at RT, and then incubated at 4°C overnight with anti-CDKN2A/p16INK4a (ab211542, 1:120; Abcam, UK) and anti-CD31 (MAB1398Z, 1:120, Millipore, US) antibodies. After being washed in PBS, the sections were incubated with Alexa Fluor 488 (1:300, Jackson ImmunoResearch, US) and Alexa Fluor 594 (1:300, Jackson ImmunoResearch, US) conjugated secondary antibodies for 2 hours and 4,6-diamidino-2-phenylindole (DAPI, 1:10,000; Sigma-Aldrich, US) for 5 minutes. Images were taken with a scanning laser confocal microscope (DMI6000B with TCS SP8 system; Leica, Wetzlar, Germany). The p16INK4a positive area was calculated with ImageJ software (ROI tool).
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!