• Donated human embryos, DPF5 or DPF6, frozen
CAUTION: experiments that involve human embryos must be conducted under IRB approval with appropriate consent and in accordance with institutional and governmental regulations
• Thaw-kit 1 (Vitrolife, cat. no. 10067)
• Mineral oil, suitable for embryo culture (e.g. Irvine Scientific cat. no. 9305)
• Acidic Tyrode’s (AT) solution (eg. Sigma-Aldrich cat. no. T1788)
• IVC1 medium (Cell Guidance Systems cat. no. M11; for recipe information, please consult Bedzhov et al, Nat. Protoc. 2014)
• IVC2 medium (Cell Guidance Systems cat. no. M12; for recipe information, please consult Bedzhov et al, Nat. Protoc. 2014)
• M2 medium (e.g. Sigma-Aldrich cat. no. M7167)
• 32% PFA (e.g. VWR cat. no. 100504-858)
• Triton X-100 10% (e.g. Sigma cat. no. 93443)
• Sodium Azide (e.g. from Sigma)
• 0.2µm filters (e.g. from Millipore, syringe, 200, and 500ml capacities)
• 10x PBS (e.g. from ThermoFisher)
• Normal Donkey Serum (e.g. Jackson ImmunoResearch cat. no. 017-000-0121)
• Primary antibodies (See "Table 1":http://www.nature.com/protocolexchange/system/uploads/4393/original/Table_1.docx?1459342868)
• Donkey anti-species whole IgG Alexa 488, 555, 594, 647-conjugated antibodies (ThermoFisher, See "Table 1":http://www.nature.com/protocolexchange/system/uploads/4393/original/Table_1.docx?1459342868)
• Donkey anti-species Fab Alexa 488, 594, 647-conjugated antibodies (Jackson ImmunoResearch, See "Table 1":http://www.nature.com/protocolexchange/system/uploads/4393/original/Table_1.docx?1459342868)
• Unlabeled Fc fragments (primary species) (Jackson ImmunoResearch, See "Table 1":http://www.nature.com/protocolexchange/system/uploads/4393/original/Table_1.docx?1459342868)
• Unlabeled Donkey anti species IgG Fab (Jackson ImmunoResearch, See "Table 1":http://www.nature.com/protocolexchange/system/uploads/4393/original/Table_1.docx?1459342868)
• DAPI (Life, D1306)
• Phalloidin-Alexa 647 (e.g. ThermoFisher cat. no. A22287)
• Glycine or Lysine powder (Sigma)
Reagent Setup
• Thawing solutions: place 500 µl of ETS1, ETS2, ETS3, and Cryo-PBS each in a well of a 4-well dish and let warm to room temperature (about 10-15 minutes)
• For removal of zona pellucida: prepare 3 dishes containing droplets (100-200 µl per droplet) of AT, M2 medium, or IVC1 medium respectively; cover the droplets with mineral oil gently pipetted in the dish to avoid disturbance of the droplets; place the IVC1 dish in the incubator until ready for use
• 8 well ibiTreat µ-slides: fill the required number of wells with 200 µl IVC1 each (we usually grow 3-4 embryos per well); if some wells are not used, fill the empty wells with 200 µl PBS
CAUTION: Depending on the microscope and objective used, the wells at the edge cannot be imaged completely, therefore it is advisable to only use the 4 wells in the center of the slide
• 2x Fix: 8% PFA in 2xPBS. To prepare, add 10 ml of 32% PFA to 8ml of 10x PBS and 22ml ddH2O. The solution should be protected from light and is stable at 4°C for 2 weeks.
• 1x Fix: prepare fresh on the day of use. Mix equal volumes of 2x Fix and ddH2O
• PBS 1x: to prepare, mix 1 volume of 10x PBS with 9 volumes of ddH2O
• Wash Buffer: to prepare, mix 1x PBS with 1/100 volumes of 10% Triton-X; filter with a 0.2µm pore size filter
IMPORTANT: Filter the buffer frequently (e.g. once a week) to clear accumulated dust or precipitate.
• Sodium Azide: prepare a 10% solution in ddH2O by mixing 10g of sodium azide in 100ml of ddH2O, and filter with a 0.2 µm pore size filter
• Glycine or Lysine: prepare 100mM Glycine or Lysine solution in PBS, and filter with a 0.2 µm pore size filter
• Quench buffer: Mix 9 volumes of wash buffer with 1 volume of 100 mM Glycine/Lysine, add Sodium Azide 1:100, and filter with a 0.2 µm pore size filter
• Block buffer: dissolve normal donkey serum in 10 ml ddH2O (the species of the serum should be matched to the species of the secondary antibodies), add 90 ml ddH2O, 10ml 10xPBS, and sodium azide 1:100. Filter with a 0.2 µm pore size filter. Use fresh or freeze aliquots at -20. Re-filter when thawing to remove solids.
IMPORTANT: Filter the buffer frequently (e.g. once a week) to clear accumulated dust or precipitate.
• DAPI: to make a 50x stock solution, dilute DAPI concentrate 1:1000 in filtered dH2O; store light protected at 4°C up to 4 months.
IMPORTANT: DAPI is insoluble in PBS and therefore dH2O must be used to dilute the stock concentrate
• DAPI 1x solution: dilute 50x DAPI stock 1:50 in Wash buffer; store light protected at 4°C up to 3 months or until signs of microbial growth
• Phalloidin-conjugates: aliquot and store at -80°C in single-use aliquots