Griffonia simplicifolia isolectin b4

Both vaso-obliteration and pathological neovascularization in the OIR mouse retinas were quantified at P17, as previously described.⁵⁷ (link) Briefly, the eyes were fixed with 4% paraformaldehyde dissolved in PBS at room temperature for 1 hour, and the retinas were dissected for overnight incubation with fluorescent Griffonia simplicifolia isolectin B₄ (Invitrogen, Thermo Fisher Scientific) and flat mounted. Images of retinal whole mounts were examined by fluorescence microscope (Zeiss AxioOberver.Z1; Carl Zeiss, Oberkochen, Germany). Vascular loss and neovascular areas in OIR were quantified by using Adobe Photoshop (Adobe Systems, San Jose, CA, USA) and ImageJ (NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/). Vascular density in the developing retinas was analyzed, using the Vessel Analysis plugin with Fiji.⁶¹ (link) Researchers performing quantification of retinal vessels were masked to the identity of samples; n represents the number of eyes quantified.⁶² (link)

The antibodies targeting GAPDH (cat. no. 10494-1-AP), CD31 (cat. no. 11265-1-AP), cyclin A1 (cat. no. 13295-1-AP), cyclin B1 (cat. no. 55004-1-AP), cyclin D1 (cat. no. 60186-1-1 g), cyclin E1 (cat. no. 11554-1-AP), CDK1 (cat. no. 19532-1-AP), CDK2 (cat. no. 10122-1-AP), poly (ADP-ribose) polymerase (PARP)1 (cat. no. 13371-1-AP), p21 (cat. no. 10355-1-AP) and p53 (cat. no. 10442-1-AP) were purchased from Proteintech Group, Inc. DMEM, fetal bovine serum (FBS), DAPI, TRIzol^®, Super Signal West Pico chemiluminescent substrate, and HRP-(cat. no. A-10677), FITC-(cat. no. A-10683) and Alexa Fluor555-conjugated secondary antibodies (cat. no. A-27017) were obtained from Thermo Fisher Scientific, Inc. Propidium iodide (PI) was obtained from Beyotime Institute of Biotechnology. The PrimeScript RT kit and SYBR Premix were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The Annexin V-FITC apoptosis kit was purchased from eBioscience (San Diego, CA, USA). Griffonia simplicifolia isolectin B4 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Matrigel Matrix was purchased from BD Transduction Laboratories (Shanghai, China). The EdU staining kit was obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Bovine serum albumin was obtained from Weiao Bio (Shanghai, China).