Immunofluorescence Staining of Hematopoietic Stem Cells

In order to prepare immunofluorescence slides of certain hematopoietic stem or progenitor cell subsets, characterized elsewhere [68 (link),107 (link)], normal BM samples were stained using following primary antibodies: Lin-FITC, CD90-PE, CD34-APC, CD38-AlexaFluor700, CD45-APC-H7, CD45RA-PE-Cy-7 or CD38-FITC, CD34-PE, CD19-APC, CD20-APC-H7 (BD Biosciences, San Jose, CA, USA). Cells of defined immunophenotype were sorted using MoFlo-XDP cell sorter (Beckman Coulter, Brea, CA, USA) into 20 mL PBS drops on poly-L-lysine coated slides. After settling, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X100. Afterwards, samples were incubated with 0.25% glycine for 30 min, followed by blocking with 3% BSA in PBS for 1 h. Subsequently, samples were stained overnight at 4 °C with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland) in a moisture chamber. After 5 washing steps, slides were incubated with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, Carlsbad, CA, USA) and DAPI for 1 h in darkness. After the next 5 washing steps, cells were analyzed using a Zeiss confocal microscope with ZEN Software (Zeiss, Oberkochen, Germany).