Alexa fluor 594 conjugated donkey anti rat igg h l secondary antibody

BMDMs and peritoneal macrophages were incubated with the fluorescent dye-conjugated antibodies for 30 min at 4 °C, briefly washed in 0.5% BSA in PBS and analyzed using FORTESSA (Beckton Dickinson) after DAPI (Molecular Probes, D3571) or Sytox blue (Molecular Probes, S34847) staining to allow exclusion of dead cells. The antibodies used were: FITC-conjugated rat anti-mouse CD11b (BD Biosciences, 553310), rat anti-mouse F4/80 (Abcam, ab6640) with Alexa Fluor 594 conjugated donkey anti-rat IgG (H + L) secondary antibody (Thermo Fisher, A-21209), and the relevant isotype controls. Ten thousand total events were collected and gated for analysis of live cells.

Pups were anesthetized under isoflurane and cerebral vessels were labeled by transcardiac infusion of DyLight 488 labeled Lycopersicon esculentum (Tomato) Lectin (50 µL, Vector Lab, Burlingame, CA, USA) 5 min before sacrifice. The animals were then perfused with ice cold heparinized PBS to remove any residual blood in cerebral vessels followed by 4% paraformaldehyde (PFA). The brains were removed, post-fixed in PFA, and dehydrated in 30% sucrose for 2 days. After immersion in Optimal cutting temperature compound (OCT), brain tissues were sectioned at a thickness of 20 µm using a Leica cryostat (Leica, Buffalo Grove, IL, USA). Brain slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) and then incubated with Alexa Fluor 594-conjugated donkey anti-rat IgG (H + L) secondary antibody (Thermo) for 1 h at room temperature. Brain slices were mounted and coverslipped using fluorescent mounting media (Dako, ‎Glostrup, Denmark). All slices were scanned with a Zeiss LSM 710 confocal microscopy (Zeiss, Oberkochen, Germany) in Advanced Imaging and Microscopy Facility of Center of Neonatal biology at Loma Linda University. Images were projected from 18 µm stacks using NIH Image J software.