Objective The collateral circulation represents an endogenous anastomotic route that compensates for the stenosis or occlusion of intracranial arteries. It supplies collateral blood flow, thereby sustaining the survival of the ischemic penumbra. This study compared the labeling of cerebral collateral arteries in mice in vitro and in vivo, using different experimental method. Methods Primary ( Willis circle ) and secondary (leptomeningeal) collateral arteries in mice were labeled in vitro using latex, DiI and ink-gelatin perfusion techniques, respectively. The diameter of the collateral arteries was then measured. Specifically, latex perfusion was employed to compare the number of leptomeningeal collaterals(LMCs) between different mouse strains. Stable in vivo observation of LMCs was achieved by constructing a closed cranial window. A mouse model of distal middle cerebral artery occlusion stroke was established via electrocoagulation, followed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Additionally, LMC diameter was continuously monitored using a fluorescence stereomicroscope, and blood flow in the peri-infarct area was detected by laser speckle contrast imaging. Results Latex and DiI perfusion both labeled the collateral arteries, but not veins, with no significant difference in vessel diameter measured by the two method ( P>0. 05 ). DiI perfusion often exhibited suboptimal perfusion efficiency. The number of LMCs was significantly higher in C57BL / 6N compared with BALB/ c mice (P<0. 000 1). The clarity and imaging quality of the cranial window were not significantly affected during the 3-week closed cranial window period. TTC staining confirmed stable construction of the mouse stroke model. LMC diameter increased and the blood flow in the peri-infarct area recovered after ischemic stroke (P<0. 05), with a strong positive correlation between them (P<0. 05). Conclusions Latex perfusion can effectively label primary and secondary collateral arteries in mice in vitro. Fluorescence stereomicroscopy and laser speckle contrast imaging can be used for continuous observations of LMCs in vivo.Remodeling of collateral vessels after ischemic stroke may promote the recovery of blood flow in the penumbra.