(a) NHS-azide was injected into the vitreous space. Twenty-four hours later, retinal and optic nerve tissue was collected for either biochemistry or immunofluorescence. Right panels: We found labeled cells across the surface of the retina after a click reaction with an Alexa-488 alkyne but did not detect label in the optic nerve. Retina scale bar, 20 µm, ON scale bar, 100 µm. Left panel: With a biotin alkyne click reaction, we were able to detect proteins in the optic nerve and show that they decreased after optic nerve crush, compared to a low background without a biotin alkyne click reaction. Bands in the control lane (right) are endogenous biotin-bound proteins. (b) Biotin-beta-alanine was injected intravitreously or provided to HEK cells in culture. Left panel: Biotinylated proteins of different masses were detectable by western blot from HEK cells. Right panels: After 24 hr, biotin-beta-alanine was detected in sections through the optic nerve, compared to optic nerve from animals with intravitreal PBS injection. Scale bar, 100 µm. Both techniques are valuable for biochemistry and histology, although protein labeling with N-hydroxysuccinimidobiotin (NHS-biotin) was superior for mass spectrometry.