Materials and Reagents

1. 0.22 μm filter (Millipore, catalog number: SLGPR33RB)

2. 75 cm² Flask (Corning, catalog number: 430641)

3. 15 mL tube (Falcon, catalog number: 352095)

4. 12 mm diameter circle coverslips (Marienfeld, catalog number: MAR0111520)

5. Glass slides (Premiere, catalog number: 9308W)

6. 24-well plate (Costar, catalog number: 3524)

7. 15 cm Petri dish (Corning, catalog number: 430599)

8. Filter paper (GE, catalog number: 10311611)

9. Parafilm (Bemis, catalog number: PM-996)

10. Milli-Q water

11. Fibronectin (1 mg/mL) (Sigma-Aldrich, catalog number: FC010), store at 4°C

12. Papain (Worthington Biochemical Corporation, LS003126), store at 4°C

13. DMEM (ThermoFisher Scientific, Gibco^TM, catalog number:12430054), store at 4°C

14. Primocin (50 mg/mL) (InvivoGen, ant-pm-2), store at -20°C

15. Fetal bovine serum (Ausbian, VS500T), store at -20°C

16. Poly-L-lysine hydrobromide (Sigma-Aldrich, catalog number: P1399), store the powder at -20°C.

    Note: Sterilize the stock solution (100 mg/mL Poly-L-lysine in Milli-Q water) with a 0.22 μm filter, and store it in aliquots at -20°C.

17. Trition X-100 (Sangon Biotech, catalog number: A110694)

18. Tween 20 (Sangon Biotech, catalog number: A100777)

19. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148), store the powder at 4°C

20. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3912), store the powder at 4°C

21. Mouse anti-Acetylated Tubulin (Sigma-Aldrich, catalog number: T6793), store at 4°C

22. Rabbit anti-Cep290 (home-made) (Zhao et al., 2021), store at -20°C

23. Guinea pig anti-Odf2 (home-made) (Zhao et al., 2019), store at -20°C

24. Donkey anti-mouse IgG conjugated with Alexa Fluor 488 (ThermoFisher Scientific, catalog number: A-21202), long-term storage at -80°C, short-term storage at 4°C

25. Donkey anti-rabbit IgG conjugated with Cy3 (Jackson ImmunoResearch, catalog number: 711-165-152), long-term storage at -80°C, short-term storage at 4°C

26. Donkey anti-guinea pig IgG conjugated with Alexa Fluor 647 (Jackson ImmunoResearch, catalog number: 706-605-148), long-term storage at -80°C, short-term storage at 4°C

27. ProLong Diamond Antifade Mountant (ThermoFisher Scientific, catalog number: P36970), store at 4°C

28. Immersion oil (Lecia microsystem, catalog number: 12847995)

29. PBS (see Recipes), store at 4°C

30. Dissection buffer (see Recipes), store at 4°C

31. Enzymatic digestion buffer (see Recipes), freshly prepared

32. mEPC culture medium (see Recipes), store at 4°C

33. Starvation culture medium (see Recipes), store at 4°C

34. Deciliation buffer (see Recipes), store at 4°C

35. 4% PFA (see Recipes)

36. TBST (10×) (see Recipes)

37. Blocking buffer (see Recipes), stored in aliquots at -20°C

Equipment

1. Milli-Q (Millipore, model: Advantage A10)

2. Sharp tweezer (Dumont, catalog number: 0208-5/45-PO)

3. Tweezer (Dumont, catalog number: 0203-5/15-PO)

4. Horizontal shaker (Zhichu, model: ZQZY-AS9)

5. Superspeed centrifuge (ThermoFisher Scientific, model: Sorvall LYNX 6000, catalog number:75006590)

6. Swinging bucket rotor (ThermoFisher Scientific, model: BIOFlex^TM HC, catalog number: 75003000)

7. Confocal microscopy (Leica, model: TCS SP8 WLL system equipped with a 63×/1.4 oil immersion objective)

Procedure