Alexa fluor plus 555 conjugated donkey anti rabbit igg antibody

Teratomas were harvested and fixed in 4% paraformaldehyde solution (Santa Cruz Biotechnology, Inc.) for 24hr at room temperature then washed with PBS 3 times and stored in PBS at 4°C until ready for use. Samples were cryoprotected with sucrose (Sigma-Aldrich), cryopreserved in Tissue-Tek O.C.T. compound (Sakura), and sectioned at 5µm with a Leica CM3050 S cryostat. Sections were incubated with PBS containing 0.1% triton X-100 (Sigma-Aldrich) and 5% donkey serum (Jackson ImmunoResearch). Sections were incubated overnight with unconjugated goat anti-mouse/rat CD47 N-terminal IgV-like extracellular domain antibody (R&D Systems), biotin-conjugated anti-mouse CD55 REAfinity antibody (clone REA300, Miltenyi Biotec), unconjugated rabbit anti-mouse CD59a antibody (clone 108, SinoBiological), and AF594-conjugated anti-human mitochondria antibody (clone 113-1, Novus Biologicals) followed by a 1hr incubation with Alexa Fluor 647-conjugated AffiniPure donkey anti-goat IgG antibody (Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), or Alexa Fluor Plus 555-conjugated donkey anti-rabbit IgG antibody (Invitrogen). Slides were mounted with ProLong Gold Antifade Mountant with DNA stain DAPI (Invitrogen) and imaged on a Zeiss LSM 800 confocal laser scanning microscope. The resulting images were processed and analyzed with Fiji (ImageJ).