Labeling details for fixed-cell imaging
| Permeabilization | Round 1 of labeling | Round 2 of labeling | |||||
|---|---|---|---|---|---|---|---|
| Blocking (min) | Primary antibody | Secondary antibody | Blocking (min) | Primary antibody | Secondary antibody | ||
| CD36 Halo-JF549/β1-integrin/CD9 | Yes After CD36 and β1-integrin Before CD9 | 30 | Anti–β1-integrin K20 (IMBULK1B; Beckman Coulter) 1:10,000 1 h | Anti–mouse-AF488 (A-11029; Thermo Fisher Scientific) 1:1,000 15 min | 30 | Anti–CD9-AF647 (sc-13118) 1:25 30 min | - |
| CD36 Halo-JF549/β1-integrin/CD81 | No | 30 | Anti–β1-integrin K20 1:100,000 1 h | Anti–mouse- AF488 1:1,000 15 min | 30 | Anti–CD81-AF647 (sc-23962) 1:300 30 min | - |
| CD36 Halo-JF549/β1-integrin/CD151 | No | 30 | Anti–β1-integrin K20 1:100,000 1 h | Anti–mouse-AF488 1:1,000 15 min | 30 | Anti–CD151-AF647 (ab199200) 1:250 30 min | - |
| CD9/CD81/CD151 | Yes After CD81 and CD151 Before CD9 | 30 | Anti–CD151-AF647 1:250 and Anti–CD81-AF488 1:100 Both for 30 min | - | 30 | Anti–CD9-AF546 1:200 30 min | - |
| CD36 Halo-JF549/CD36 Halo-JF650/anti-CD36 FA6-152 Note: CD36 Halo-JF650 not used in analysis | No | 15 | Anti–CD36 FA6-152 1:400 30 min | Anti–mouse-AF488 1:1,000 15 min | - | - | - |
| CD36 Halo-JF549/anti-CD36 FA6-152/caveolin-1 Note: Anti-CD36 FA6-152 not used in analysis | Yes After CD36-Halo and anti-CD36 FA6-152 Before caveolin-1 | 15 | Anti-CD36 FA6-152 (ab17044) 1:400 30 min | Anti–mouse-AF488 1:1,000 15 min | 30 | Anti-caveolin-1 (ab32577) 1:200 30 min | Anti–Rabbit-AF647 (Invitrogen, A32733) 1:1,000 15 min |
| CD36 Halo-JF549/pSFK(Y419)/Fyn | Yes After CD36 Before pSFK(Y419) and Fyn | 15 | Anti–phospho-Src (Tyr419)-AF488 (44–660A1; Thermo Fisher Scientific) 1:500 & Anti-Fyn (ab1881) 1:400 Both for 1 h | Anti–mouse-AF647 (A21236; Thermo Fisher Scientific) 1:1,000 15 min | - | - | - |
| CD36 Halo-JF549/caveolin-1/phalloidin Note: Only phalloidin channel used from this dataset | Yes After CD36 Before caveolin-1 and phalloidin | 30 | Anti-caveolin-1 1:200 30 min | Anti–Rabbit-AF647 1:1,000 15 min | 15 | Phalloidin-AF647 (A22287; Thermo Fisher Scientific) 1:20 15 min | |
| CD36 Halo-JF549/active β1-integrin/CD9 Note: CD9 not used in analysis | Yes After CD36 and active β1-integrin Before CD9 | 60 | Anti- active β1-integrin 12G10 (ab30394; Abcam) 1:500 1 h | Anti–mouse-AF488 1:1,000 15 min | 30 | Anti–CD9-AF647 1:25 30 min | |
| CD36 Halo-JF549/β1-integrin/phalloidin | Yes After CD36 and β1-integrin Before phalloidin | 30 | Anti–β1-integrin K20 1:100,000 1 h | Anti–Rabbit-AF488 1:1,000 15 min | 15 | Phalloidin-AF647 1:200 15 min | |
| CD36 Halo-JF549/CD9/phalloidin | Yes After CD36 Before CD9 and phalloidin | 30 | Anti–CD9-AF647 1:25 and Phalloidin-AF488 (A12379; Thermo Fisher Scientific) 1:500 30 min | ||||
| Permeabilization | Round 1 of labeling | Round 2 of labeling | |||||
|---|---|---|---|---|---|---|---|
| Blocking (min) | Primary antibody | Secondary antibody | Blocking (min) | Primary antibody | Secondary antibody | ||
| CD36 Halo-JF549/β1-integrin/CD9 | Yes | 30 | Anti–β1-integrin K20 (IMBULK1B; Beckman Coulter) | Anti–mouse-AF488 (A-11029; Thermo Fisher Scientific) | 30 | Anti–CD9-AF647 (sc-13118) | - |
| CD36 Halo-JF549/β1-integrin/CD81 | No | 30 | Anti–β1-integrin K20 | Anti–mouse- AF488 | 30 | Anti–CD81-AF647 (sc-23962) | - |
| CD36 Halo-JF549/β1-integrin/CD151 | No | 30 | Anti–β1-integrin K20 | Anti–mouse-AF488 | 30 | Anti–CD151-AF647 (ab199200) | - |
| CD9/CD81/CD151 | Yes | 30 | Anti–CD151-AF647 | - | 30 | Anti–CD9-AF546 | - |
| CD36 Halo-JF549/CD36 Halo-JF650/anti-CD36 FA6-152 | No | 15 | Anti–CD36 FA6-152 | Anti–mouse-AF488 | - | - | - |
| CD36 Halo-JF549/anti-CD36 FA6-152/caveolin-1 | Yes | 15 | Anti-CD36 FA6-152 (ab17044) | Anti–mouse-AF488 | 30 | Anti-caveolin-1 (ab32577) | Anti–Rabbit-AF647 (Invitrogen, A32733) |
| CD36 Halo-JF549/pSFK(Y419)/Fyn | Yes | 15 | Anti–phospho-Src (Tyr419)-AF488 | Anti–mouse-AF647 (A21236; Thermo Fisher Scientific) | - | - | - |
| CD36 Halo-JF549/caveolin-1/phalloidin | Yes | 30 | Anti-caveolin-1 | Anti–Rabbit-AF647 | 15 | Phalloidin-AF647 (A22287; Thermo Fisher Scientific) | |
| CD36 Halo-JF549/active β1-integrin/CD9 | Yes | 60 | Anti- active β1-integrin 12G10 (ab30394; Abcam) | Anti–mouse-AF488 | 30 | Anti–CD9-AF647 | |
| CD36 Halo-JF549/β1-integrin/phalloidin | Yes | 30 | Anti–β1-integrin K20 | Anti–Rabbit-AF488 | 15 | Phalloidin-AF647 | |
| CD36 Halo-JF549/CD9/phalloidin | Yes | 30 | Anti–CD9-AF647 | ||||
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