Discovery v 12 microscope

For whole mount LacZ reporter staining (⁴⁴ with some adjustments), E11.5 mouse embryos were dissected in cold PBS, fixed in 4% paraformaldehyde (PFA) in PBS on ice for 30 min, followed by 3x washing with the LacZ buffer (2 mM MgCl₂, 0.01% sodium deoxycholate, 0.02% Nonidet-40 in PBS). The embryos were then incubated in staining solution (0.5 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide in LacZ buffer) at 37°C for a few hours to overnight until the desired staining was obtained. After staining, embryos were washed in LacZ buffer and imaged using Zeiss Discovery V.12 microscope and Leica DFC420 digital camera. Embryos were stored at 4°C in 4% PFA in PBS.

E18.5 foetuses were processed and stained for bone and cartilage as described previously ⁴⁵ . Foetuses were kept in H₂O for 1–2 hours at RT and heat shocked at 65°C for 1 minute. The skin was taken off and the abdominal and thoracic viscera were removed using forceps. The foetuses were then fixed in 100% ethanol overnight. The next day, the cartilage was stained overnight using alcian blue staining solution (150 mg/l alcian blue 8 GX in 80% ethanol and 20% acetic acid). On the third day, foetuses were rinsed and post-fixed in 100% ethanol overnight. On the fourth day, initial clearing was done by incubating the foetuses for 20 minutes in 1% KOH in H₂O, followed by alizarin red (50mg/l alizarin red S in 0.2% KOH) staining of bones overnight. On the fifth day and onwards rinsing and clearing is done using low concentrations of KOH. The stained embryos were dissected in 80% glycerol and limbs were imaged using Zeiss Discovery V.12 microscope and Leica DFC420 digital camera.