Lms900 confocal microscope

Immunofluorescence staining of cryosections or oocytes was performed as described previously.¹⁹ Briefly, sections or oocytes were fixed in 4% paraformaldehyde in PBS for 20 min and washed with PBS. They were then permeabilized with 0.1% Tween‐20 in PBS for 20 min and washed with PBS. The specimens were blocked with 2% bovine serum albumin (BSA) in PBS for 1 h at 25°C, followed by incubation with indicated primary antibodies in 2% BSA/PBS for 2 h at 25°C. After washing in 2% BSA/PBS for 5 min, the sections were incubated with Alexa Fluor 488‐conjugated secondary antibody (1:250; Thermo Fisher Scientific, Waltham, MA, USA) in 2% BSA/PBS for 40 min. The sections were washed and counterstained with TOPRO^TM‐3‐iodide (1:100; T3605; Thermo Fisher Scientific).
The primary antibodies used were anti‐Histone H3 (rabbit polyclonal, ab183626; Abcam, Cambridge, UK), anti‐Cep55 (rabbit polyclonal, ab170414; Abcam, Cambridge, UK), anti‐mouse vasa homolog (MVH) (rabbit polyclonal, ab13840; Abcam, Cambridge, UK), anti‐EEA‐1 (rabbit polyclonal, 2411S; Cell Signaling Technologies, Danvers, MA), anti‐Rab7 (rabbit monoclonal, 9367; Cell Signaling Technologies), and anti‐Lamp‐1 (rat monoclonal, NB100‐77683; Novus Biologicals, Littleton, CO, USA). Images were obtained using a Zeiss LMS900 confocal microscope (Oberkochen, Germany) and analyzed using the ZEN software (Zeiss).

Oocytes were stained with CellMask^TM Plasma Membrane Stain (2.5 μg/ml; C10046; Life Technologies, Carlsbad, CA, USA), Sytox^TM Green Nucleic Acid Stain (2.5 μM; S7020; Invitrogen, Waltham, MA), or ER‐Tracker^TM Red dye (1:1000; E34250; Thermo Fisher). The oocytes were washed three times in an M16 medium and observed by confocal live imaging using a Zeiss LMS900 confocal microscope.