Biolane hti 16vx

In-situ probes and tissue were prepared as described previously (Quigley et al., 2004 (link)). Probes were hybridized for 24 hr at 66°C. Post-hybridization washes were performed using a BioLane HTI 16Vx (Intavis Bioanalytical Instruments), with the following parameters: 2x SSCT 3 × 5 min, 11 × 10 min at 66°C; 0.2x SSCT 10 × 10 min; blocking solution [5% normal goat serum (Invitrogen), 2 mg/mL BSA (RPI) in PBST] for 24 hr at 4°C; anti-Dig-AP, Fab fragments (1:5000 in blocking solution, Millipore-Sigma) for 24 hr at 4°C; PBST 59 × 20 min. AP staining was performed as described (Quigley et al., 2004 (link)). Tissue for sectioning was equilibrated into 5% gelatin (300-bloom, type-A, Sigma), post-fixed in 4% PFA/PBS overnight at 4°C and sectioned on a Vibratome 1500 (Harvard Apparatus).

In situ hybridization (ISH) probes and tissue were prepared as described (Quigley et al., 2004 (link)). Probes were hybridized for 24 hr at 66°C. Post-hybridization washes were performed using a BioLane HTI 16Vx (Intavis Bioanalytical Instruments), with the following parameters: 2x SSCT 3 × 5 min, 11 × 10 min at 66°C; 0.2x SSCT 10 × 10 min; blocking solution [5% normal goat serum (Invitrogen), 2 mg/mL BSA (RPI) in PBST] for 24 hr at 4°C; anti-Dig-AP, Fab fragments (1:5000 in blocking solution, Millipore-Sigma) for 24 hr at 4°C; PBST 59 × 20 min. AP staining was performed as described (Quigley et al., 2004 (link)).