X. laevis embryos were injected with morpholinos and/or mRNAs into one blastomere at the 1-, 2-, or 16-cell stage and collected at different stages; staging was according to Nieuwkoop and Faber (27). For histologic analysis, embryos were embedded into Paraplast, and 12-μm sections were cut on a rotary microtome and stained with hematoxylin and eosin. Immunohistochemistry for Bap1 was performed as previously described (28) using an antibody raised against human BAP1 (H300; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution. Embryos were incubated with the primary antibody for 24 hours and then incubated with alkaline phosphatase (AP)–conjugated secondary antibody (ab6729, Abcam, Cambridge, MA). Color was developed using a combination of nitro blue tetrazolium (NBT) chloride and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Promega, Madison, WI). WISH was performed as described (29). Embryos were fixed in 4% MEMFA [100 mM MOPS (pH 7.4), 2 mM EGTA, and 1 mM MgSO4, and 3.7% (v/v) formaldehyde] 1 hour at RT, dehydrated with 100% methanol in three 10-min washes, and either kept at −20°C in methanol or rehydrated through a methanol wash series {75, 50, and 25% in PTw [phosphate-buffered saline (PBS) buffer and Tween 20]} for 5 min each step, followed by a 5-min PTw wash. Embryos were rinsed for 5 min, prehybridized with hybridization buffer at 65°C for 5 to 6 hours, and then hybridized overnight in hybridization buffer containing the appropriate amount of antisense probe at 65°C. Next, embryos were sequentially washed in 50% formamide/2× SSC and 0.1% Tween 20 (10 min/60°C), 25% formamide/2× SSC and 0.1% Tween 20 (10 min/60°C), 12.5% formamide/2× SSC and 0.1% Tween 20 (10 min/60°C), 2× SSC and 0.1% Tween 20 (10 min/60°C), and 0.2× SSC and 0.1% Tween 20 (30 min/60°C). After three 5-min washes with PTw and two 5-min washes with maleic acid buffer (MAB), embryos were placed in MAB/2% Boehringer Blocking Reagent (BMB) blocking reagent (Roche) for 2 hours and then incubated in MAB/2% BMB containing 1:3000 diluted anti-digoxigenin/AP (Roche, Basel, Switzerland) overnight. After incubation, embryos were washed six times with MAB for 30 min each and twice with fresh AP buffer for 5 min, transferred to a precooled color reaction solution (AP buffer containing NBT chloride and BCIP), and incubated at RT in the dark until sufficient staining was reached. The staining reaction was stopped by replacing the staining solution with methanol, twice for 5 min. Embryos were left in a third methanol wash for 40 min at 62°C and then washed three to five times with water for rehydration. To bleach pigments, embryos were treated with 2% H2O2 in 1× PBS (stocks: 30% H2O2 and PBS 20×) under fluorescent light for 4 to 6 hours, washed several times with water, and stored in 4% formaldehyde in 1× PBS at 4°C. In all experiments in which embryos were injected past the one-cell stage, fluorescein isothiocyanate was coinjected to mark the injected side.

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