Retinal Immunostaining of Mouse Embryos

Staged mouse embryos were dissected and immediately fixed in 4% paraformaldehyde (PFA) in PBS at 4°C for 2–3 hours. Samples were embedded and frozen in OCT medium (Tissue-Tek) after dehydration in graded sucrose and sectioned at 14 µm thickness. Before adult retina samples were harvested, vascular perfusion was performed to eliminate the blood remain in the retinal vessels, and then retinas were dissected and fixed in 4%PFA. Retinal flat mount immunostaining was performed as previously described [52] (link). Dilution and sources of antibodies used in this study were: mouse anti-POU4F1 (1∶500, Santa Cruz), goat anti-POU4F2 (1∶500, Santa Cruz), mouse anti-Calbindin (1∶5000, Sigma), rabbit anti-Calretinin (1∶2000, Oncogene), rabbit anti-CHAT (1∶200, Chemicon), sheep anti-CHX10 (1∶200, Exalpha), mouse anti-PAX6 (1∶200, DSHB). Mouse anti-Cyclin D3 (1∶100, Santa Cruz). Alexa-conjugated secondary antibodies (Molecular Probes) were used at a dilution of 1∶1,000. Images were captured with a Zeiss 510 META confocal microscope.

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Huang L., Hu F., Xie X., Harder J., Fernandes K., Zeng X.Y., Libby R, & Gan L. (2014). Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice. PLoS ONE, 9(4), e94173.

Publication 2014

mouse anti-POU4F1 goat anti-POU4F2 mouse anti-Calbindin rabbit anti-CHAT Mouse anti-Cyclin D3 Alexa-conjugated secondary antibodies 510 META confocal microscope

Adult Antibodies Blood Calbindin Calretinin Confocal microscope Cyclin d3 Dehydration Dilution Embryos Frozen Goat Molecular probes Mouse Oncogene Paraformaldehyde Perfusion Rabbit Retinal Retinal vessels Sheep Sucrose Tissue Vascular

Corresponding Organization : Hangzhou Normal University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of POU4F1, POU4F2, Calbindin, Calretinin, CHAT, CHX10, PAX6, and Cyclin D3 in mouse embryos and adult retina

control variables

Tissue fixation in 4% paraformaldehyde (PFA) in PBS at 4°C for 2-3 hours
Dehydration in graded sucrose and embedding in OCT medium
Sectioning thickness of 14 μm
Vascular perfusion to eliminate blood in adult retina samples
Retinal flat mount immunostaining protocol as previously described [52]
Antibody concentrations for immunostaining

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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