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2 protocols using rabbit anti β tubulin 3

1

Immunofluorescence Staining of Cryosectioned Eyes

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Eyes were harvested and fixed in 4% PFA for 2 hours prior to cryoprotection in 25% sucrose-PBS overnight. Eyes were embedded in OCT, and cryosectioned at 12 μm and mounted on SuperFrost Plus slides prior to immunofluorescence. Standard immunofluorescence procedures were followed.9 (link) Primary antibodies include: chicken anti-β-gal (Abcam Cambridge, MA; ab9361), rabbit anti-β-tubulin III (Covance, Princeton, NJ; PRB-435P), rabbit anti-Brn3 (Santa Cruz Biotechnology, Santa Cruz, CA; sc28595), and rabbit anti-Sox2 (Abcam; ab97959). Secondary antibodies used were (all from Invitrogen): chicken anti-rabbit Alexa-594, goat anti-chicken Alexa-488, goat anti-chicken Alexa-594, goat anti-mouse Alexa-488, goat anti-mouse Alexa-594, goat anti-rabbit Alexa-488, and goat anti-rabbit Alexa-594. Nuclei were stained using 1 μg/ml of Hoechst 33342 (Sigma, St Louis, MO; 14533). Slides were mounted with Prolong Gold (Invitrogen) antifade reagent and imaged using an Olympus BX61 microscope and the InVivo software (Olympus), or alternatively using confocal laser scanning microscopy with a Leica SP5 II microscope (Leica Microsystems) and LAF software (Leica Microsystems).
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2

Immunohistochemistry of Retinal Tissues

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Eyes were harvested and fixed in 4% PFA for 2 hours prior to cryoprotection in 25% sucrose-PBS overnight. Eyes were embedded in OCT, and cryosectioned at 12 μm and mounted on SuperFrost Plus slides prior to immunofluorescence. Standard immunofluorescence procedures were followed.9 (link) Primary antibodies include: chicken anti-β-gal (Abcam Cambridge, MA, ab9361), rabbit anti-β-tubulin III (Covance, Princeton, NJ, PRB-435P), rabbit anti-Brn3 (Santa Cruz Biotechnology, Santa Cruz, CA, sc28595), and rabbit anti-Sox2 (Abcam, ab97959). Secondary antibodies used were (Invitrogen): chicken anti-rabbit Alexa-594, goat anti-chicken Alexa-488, goat anti-chicken Alexa-594, goat anti-mouse Alexa-488, goat anti-mouse Alexa-594, goat anti-rabbit Alexa-488, goat anti-rabbit Alexa-594. Nuclei were stained using 1 μg/mL of Hoechst 33342 (Sigma, St. Louis, MO, 14533). Slides were mounted with Prolong Gold (Invitrogen) anti-fade reagent and imaged using an Olympus BX61 microscope and the InVivo software, or alternatively using confocal laser scanning microscopy with a Leica SP5 II microscope and LAF software.
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