Alexa 488 conjugated ctb

Mice were anesthetized with sodium pentobarbital before ONI. Optic nerves were exposed intraorbitally and crushed at about 0.5–1.0 mm from the posterior pole of the eyeball, with fine surgical forceps for 5 s.^{6 (link), 22 (link)} On d11 after ONI, 2 μg of Alexa 488-conjugated CTB (Invitrogen, Carlsbad, CA, USA) was injected into the vitreous body. On d14 after ONI, the animals were perfused with Zamboni's Fixative (2% paraformaldehyde and 15% picric acid in 0.1 M phosphate buffer). The optic nerve was removed, postfixed and immersed in 30% sucrose overnight at 4 °C. The optic nerve was then embedded in an OCT compound (Sakura, Tokyo, Japan), frozen on dry ice and 10-μm serial cross-sections were prepared using a cryostat and collected on MAS-coated glass slides (Matsunami, Osaka, Japan). To estimate the total number of regenerating axons, axonal outgrowth was quantified by counting CTB-positive axons that crossed a virtual line parallel to the lesion site at 250 and 500 μm distal to the lesion site.^{23 (link)}

BGC-823 and SGC-7901 cell lines were cultured in 24-well plates at 4 × 10⁵ cells per well. The cells after indicated treatment were gently washed with PBS, fixed in 4% paraformaldehyde (PFA) for 30 min, permeabilized by 0.1% TritonX-100, and blocked by 2% BSA. Cells were incubated with mouse anti-measles phosphoprotein (MV-P, abcam, ab43820), followed by Alexa Fluor^® 594 donkey anti-mouse IgG (H + L) (Invitrogen, A-21203) and DAPI (Beyotime, C1005) in the dark, images were captured using Zeiss cLSM780 microscope. For co-localization analysis, cells were stained with Alexa 488-conjugated CTB (Invitrogen, C-34775) for 2 h at 37 °C incubator before fixation by 4% PFA. Then cells were incubated with anti-Acid sphingomyelinase (ASMase, abcam, ab83354) at 4 °C overnight, followed by Alexa Fluor^®⁠ 594 goat anti-rabbit IgG (H + L) (Invitrogen, A-11012) and DAPI in the dark. Images were captured using Lecia TCS SP8 confocal laser scanning microscope.