Anti cd4

Fourteen days after the grafting procedure, 4 eyes receiving grafts in each group were enucleated and fixed in 10% formaldehyde solution. The samples were embedded in paraffin for preparation of 3 microns paraffin-embodied sectioned. Then, slides with sections were deparaffinized performed with hematoxylin and eosin (H&E) staining for histological examination. For immunohistological analysis, the slides were also deparaffinized and then processed for immunohistochemical staining with anti-CD4(Sigma) and anti-vWF(Sigma) according to the previous reported [17] (link) antibodies. Briefly, after deparaffinization, the slides were rehydrated and heat-induced antigen retrieval was performed. After blocking with 10% normal horse serum for 15 min, the sections were immunostainned using the primary antibodies and incubated overnight at 4°C. The redundant antibodies were washed with Phosphate Buffered Saline. The corresponding secondary antibodies were added and incubated for 2 hours at room temperature.

Whole BM cells were collected by flushing femurs from donor GFP transgenic mice with phosphate-buffered saline (PBS). Recipient C57BL/6 mice were treated intraperitoneally with 25 mg/kg of busulfan (Cat. #B2635; Sigma, St. Louis, MO, USA) 4 days before BMT and with 30 µg of anti-CD4 (Clone GK1.5, Cat. #BE0003-1, Bio-X-Cell, Lebanon, NH, USA) and anti-CD8 (Clone 2.43, Cat. #BE0061, Bio-X-Cell) antibodies 2 days before BMT. Treated mice were then injected intravenously with 5 × 10⁶ GFP⁺ BM cells. After allowing 4 weeks for BM cell engraftment, the transplant efficiency was confirmed by evaluating GFP⁺ BM cells collected from at least 3 representative transplanted mice using flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA; Figure S1).