Fluorescent Immunohistochemistry of Brain Tissue

Fluorescent immunohistochemistry (IHC) was performed on 20-μm cryosectioned PFA-fixed brain tissue as described in refs. 67 (link) and 84 (link)–87 (link, link, link). Primary antibodies were diluted in blocking buffer and incubated on tissue sections overnight at 4 °C. The following antibodies and dilutions were used: mouse anti-Brn3a (diluted 1:125, Millipore), rabbit anti-RFP (diluted 1:500, Rockland), rabbit anti-Opn4 [diluted 1:2,000, Dr. C.K. Chen’s laboratory (67 (link))], mouse anti- SMI32 (diluted 1:1,000, Covance), rabbit anti-RBPMS (diluted 1:500, PhosphoSolutions), rabbit anti-GFP (diluted 1:250, Invitrogen), mouse anti-NeuN (diluted 1:200, Millipore), rabbit anti-GFAP (1:1,000, DAkoCytomation), rabbit anti-Iba1 (1:500, Wako), mouse anti-GAD67 (diluted 1:500, Millipore), rabbit anti-calbindin (diluted 1:2,500, Swant), rabbit anti-calretinin (diluted 1:2,000, Swant), mouse anti-synaptophysin (diluted 1:500, SySy), and goat anti-NPNT (diluted 1:40, R&D systems). A minimum of three animals (per genotype and per age) were compared in all IHC experiments. Please reference SI Appendix for more details.

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Su J., Sabbagh U., Liang Y., Olejníková L., Dixon K.G., Russell A.L., Chen J., Pan Y.A., Triplett J.W, & Fox M.A. (2021). A cell–ECM mechanism for connecting the ipsilateral eye to the brain. Proceedings of the National Academy of Sciences of the United States of America, 118(42), e2104343118.

Publication 2021

mouse anti-Brn3a rabbit anti-RFP mouse anti- SMI32 rabbit anti-RBPMS rabbit anti-GFP mouse anti-NeuN rabbit anti-GFAP rabbit anti-Iba1 mouse anti-GAD67 rabbit anti-calbindin rabbit anti-calretinin

Animals Anti synaptophysin Antibodies Brain Buffer Calbindin Calretinin Dilutions Gad67 Genotype Gfap Goat Immunohistochemistry Mouse Rabbit Rbpms Tissue

Corresponding Organization : Carilion Clinic

Other organizations : Columbia University, National Institute of Mental Health, Children's National, George Washington University, Virginia–Maryland College of Veterinary Medicine

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Variable analysis

independent variables

Genotype

dependent variables

Immunofluorescence staining patterns of various markers (Brn3a, RFP, Opn4, SMI32, RBPMS, GFP, NeuN, GFAP, Iba1, GAD67, calbindin, calretinin, synaptophysin, NPNT) in brain tissue sections

control variables

20-μm cryosectioned PFA-fixed brain tissue
Primary antibody dilutions
Incubation conditions (overnight at 4 °C)

controls

Positive controls: None specified
Negative controls: None specified

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