Alexa fluor 488 conjugated anti tomm20 antibody

SH-SY5Y cells were seeded onto an 8-well culture slide (Invitrogen) at 1×10⁴ cells per well, incubated at 37°C, 5% CO₂ for 24 h, fixed with 4% PFA in DPBS at RT for 10 min, blocked with Blocking One Histo (Nacalai) at RT for 10 min, stained with anti-RD3 antibody (Santa Cruz, sc-390653, 1:100) at 4°C overnight, washed twice, stained with anti-mouse Alexa Fluor 647-conjugated secondary antibody (Invitrogen, A-31571, 1:400) at RT for 2 h, washed three times, stained with Alexa Fluor 488-conjugated anti-Tomm20 antibody (Abcam, ab205486, 1:500) and DAPI (Dojindo, 1:1,000) at RT for 1 h, washed twice, mounted with FluorSave (Merck Millipore), incubated at 4°C overnight, and stored at 4°C until imaging by Nikon Ti Eclipse Confocal Microscope.

Mitochondria distribution imaging was performed, following Onodera et al.¹⁰⁴ (link) First, SH-SY5Y cells (RD3 wildtype and knockout cells) were seeded onto an 8-well culture slide (Invitrogen) at 5×10³ cells (for Control) and 1×10⁴ cells (for Cilium Induction) per well, and incubated at 37°C, 5% CO₂ for 24 h. Next, ciliogenesis was induced by changing the media for fresh serum-free media, incubated at 37°C, 5% CO₂ for 24 h, fixed by adding formaldehyde (final: 2%) in DPBS at 37°C for 10 min, washed twice, permeabilized with chilled absolute MeOH on ice for 5 min, washed twice, stained with anti-α-Tubulin antibody (Santa Cruz, sc-32293, 1:250) at 4°C overnight, washed twice, stained with anti-mouse Alexa Fluor 594-conjugated secondary antibody (Invitrogen, 1:400) at RT for 2 h, washed three times, stained with Alexa Fluor 488-conjugated anti-Tomm20 antibody (Abcam, ab205486, 1:500), Alexa Fluor 647-conjugated anti-γ-Tubulin antibody (Abcam, ab191114, 1:500) and DAPI (Dojindo, 1:1,000) at RT for 1 h, washed twice, mounted with FluorSave (Merck Millipore), then stored at 4°C until imaging by Nikon Ti Eclipse Confocal Microscope.