Immunofluorescence staining techniques for retinal stretched preparation in mice

Objective To search for a simple and easily handled immunofluorescence technique for retinal stretched preparation in mice.Methods A total of 30 C57BL/6J adult mice were perfused with 40 g·L-1 paraformaldehyde through the heart.Eyeballs were enucleated and retinas were removed.All mice were divided randomly into 3 groups.Twenty retinas in group A were treated with 3 g·L-1 Triton X-100 at 4 ℃ for 1 hour;Twenty retinas in group B were firstly put into methanol for 30 minutes,and then treated with 3 g·L-1Triton X-100 at 4 ℃ for 1 hour;Twenty retinas in group C were firstly put into methanol for 30 minutes,and then treated with 3 g·L-1 Triton X-100 for staying overnight at 4 ℃;a-smooth muscle actin(a-SMA)immunofluorescence staining was used,and photograph was taken under fluorescence microscope in all groups.The difference was observed.Results Under fluorescence microscope,morphologies of retinal vessels were with weak fluorescence and fuzzy circumscription in group A;Retinas were with fluorescence,vascular branches were seen clearly but with weak fluorescence in group B;Retinas were with strong fluorescence,a-SMA was found in cell membranes of vascular endothelial cells and perithelial cells,with vacuolus and light background staining.Conclusion Retinal stretched preparation immunofluorescence is a better,simple and reliable way to observe morphologies of retinal tissues.In the experiment,the key for successful staining is to defat with methanol and soak with enough Triton X-100.