Rabbit anti human cd34 antibody

CD34 + HSPCs either transduced with nef-expressing lentiviruses or infected with NL43 were transferred to a slide by cytospin. IF was performed with a standard protocol. For nef (zsgreen1) expression slides were stained with a rabbit anti-human CD34 antibody (Abcam) and an Alexfluor 555 conjugated second antibody (Thermal fisher). For intracellular P24 expression slides were stained with a mouse anti-P24 antibody (Immunoway) and an Alexfluor 488 conjugated second antibody (Thermal fisher) after surface staining for CD34. All the slides were finally counter-stained with DAPI and photomicrographs were taken with an Olympus confocal microscopy with a 100 × oil objective.

Frozen scaffold blocks were cut into 10–30 μm thickness using a cryostat (Leica Biosystems) and stored at −80 °C until use. For hematoxylin and eosin, and Masson’s Trichrome staining, frozen tissue sections were fixed with 10 % buffered formalin solution and stained following the vendor’s protocol (American Master Tech). For immunofluorescence staining, frozen tissue sections were fixed with ice-cold acetone, blocked with 10 % normal goat serum and 1 % bovine serum albumin diluted in phosphate buffered saline (PBS). Slides were incubated with rat anti-mouse CD31 (BD Pharmingen) and rabbit anti-human CD34 antibody (Abcam) for overnight. Subsequently the slides were stained by goat anti-rat immunoglobulin G (IgG) conjugated with alexa fluor 488 and goat anti-rabbit IgG conjugated with alexa fluor 568 (Invitrogen) for 1 h. Finally, VectaShield mounting medium with DAPI was applied and slides were imaged under a fluorescence (Zeiss 200) microscope. Open source image analysis software, ImageJ, was used to process and quantify areas of collagen fibers and vasculatures in Masson’s Trichrome and mouse CD31 stained slides, respectively.