Laser dissection microscope

Drops of worms fixed in 70% methanol were pipetted onto a Leica PEN membrane slide (76 × 26 mm P/N 11505158). The slide was then placed on a slide warmer at 50°C to evaporate the methanol. Once dried, the slide was mounted onto a Leica laser dissection microscope. Costar Thermowell tubes (0.5 ml with flat cap, Cat#: 6530) were loaded into the collection holder, and 65μL of lysis buffer (Ambion RNAqueous-Micro Kit, ThermoFisher Scientific, cat # AM1931) was pipetted into the tube cap. Using the 40x objective, 50–100 tail tips were dissected into each cap. Before unloading the tube from the collection holder, an additional 35μL of lysis buffer was added to the tube cap. Tubes were spun down, flash frozen in liquid nitrogen or on dry ice and stored at −80 °C until enough tissue was collected for one RNA extraction (240–440 tail tips). RNA was extracted using the Ambion RNAqueous-Micro Kit following the manufacturer’s protocol and eluted twice into 8 μl elution solution. The quality and quantity of the extracted RNA was evaluated with Agilent high sensitivity RNA screen tapes (#5067–5579).

Hsp60^flox/WT X VillinCre^Tg mice were mated with Hsp60^flox/flox mice. When females were plug positive, embryos were assumed to be in developmental stage E 0.5 (0.5 days post coitum). At the indicated stage (E12.5–13.5) dams were killed by cervical dislocation and the uterus was removed. The embryos and the surrounding amniotic sac (visceral endoderm) were prepared from the uterus, examined under the microscope, killed and tails were taken for genotyping.
For genotyping, tail cuts or ear punches were lysed in a 10 mM Tris-HCl buffer pH 8.0 buffer containing 50 mM KCl, 0.45% Nonidet P40, 0.45% Tween 20 and 0.5 mg ml⁻¹ Proteinase K overnight (O.N.) at 65 °C and inactivated at 95 °C for 10 min. Two microlitre of the clear supernatant was used for Crimson-Taq PCR (NEB, Ipswich, MA). For genotyping of jejunal villi and crypts of Hsp60^Δ/ΔIEC mice 10 μm thick cryosections were H&E stained (Harris formulation), 50,000 μm² cells were cut using the laser-dissection microscope (Leica, Soest, Germany) and lysed in RLT Plus buffer (Qiagen, Hilden, Germany). DNA was isolated using the Allprep DNA/RNA Mini kit (Qiagen). Two microlitre of DNA were used for Crimson-Taq PCR. Primers used for genotyping are given in Supplementary Table 1. All uncropped agarose gels can be found in Supplementary Fig. 11.