Mouse anti dig

Immunohistochemistry experiments were performed as previously described70 (link). In brief, larval CNSs were dissected in PBS, fixed in 4% formaldehyde. Washing steps were performed using PBS with 0.3% Triton X-100 (3 × 5 min) and primary antibodies were incubated overnight at 4 °C. Antibodies used were guinea pig anti-Deadpan (1:1,000, kind gift from Jürgen Knoblich), mouse anti-Pros (1:100, MR1A, Developmental Studies Hybridoma Bank, DSHB), mouse anti-Repo (1:100, 8D12, DSHB), mouse anti-Dlg (1:20, DSHB), mouse anti-pH3 (1:1,000, Cell Signaling Technology), mouse anti-β-Gal (1:375, Promega), mouse anti-Dig (1:1,000, Roche), anti-Echinoid (1:1,000, kind gift from Laura Nilson), rabbit anti-Yorkie (1:400, kind gift from Kenneth Irvine), rabbit anti-Expanded (1:1,000, kind gift from Richard Fehon).
In situ probe for crumbs was PCR-generated using the following forward primers: 5′-CGTTGGTGGCCAGAAATTGG-3′ and 5′-CACAGTGCTGACCCTCGAAT-3′ (5′-TAATACGACTCACTATAGGAGACCAC-3′) as reverse primer ((XX)=T7 sequence). After purification of the PCR product the in vitro transcription using T7 and the Dig RNA Labelling Mix (Roche) was used to generate the RNA in situ probe.

Dissected ovaries were fixed in 4% PFA/PBS for 20 min at room temperature. After fixation and washes in PBST (0.2% Tween 20/PBS), 3 μg/ml proteinase K was applied for 12 min at room temperature. The digestion was stopped with 2 mg/ml glycine. The samples were washed twice with PBST and were refixed with 4% PFA/PBS for 20 min at room temperature. After washes in PBST, PBST was gradually replaced with hybridization buffer (50% formamide, 5×SSC (pH 5.0), 0.1 mg/ml salmon sperm DNA, 50 μg/ml heparin, 0.1% Tween 20), and the samples were prehybridized for 1 h at 55°C. Hybridization was carried out at 55°C overnight in hybridization buffer with RNA probes for osk, bcd, or grk synthesized in the presence of digoxigenin (DIG)-UTP (Roche, Penzberg, Germany). Excess probes were removed by 3 washes with 0.1% Tween 20/50% formamide/5×SSC for 10 min each at 55°C followed by 4 washes with 0.1% Tween 20/50% formamide/2×SSC for 30 min each at 55°C. Then, the wash buffer was gradually replaced with PBST. The hybridized DIG-labeled probes were detected with standard procedures using mouse anti-DIG (Roche, 1: 400) and Alexa-488-conjugated anti-mouse IgG (Thermo Fisher Scientific, 1: 500).