When data for all six PEEP levels were collected, the animal was returned to the supine position with intra-abdominal pressure normal (if not already) for 5 min before proceeding to the next randomly selected condition.
For Calretinin immunostaining, explants were pretreated with 0.01% pronase for 30 min before proceeding with the normal horse serum blocking.
Participants took a short rest of 2 3 min before proceeding to the next block.
After the incubation period, the plates were allowed to equilibrate at room temperature for 30 min before proceeding to develop the luminescent signal.
For the DNAase treated samples in Figure 4 figure supplement 1, benzonase (25U) was added to the extracts before incubating at room temperature for 30 min before proceeding with immunoprecipitation.
Where this is not possible, the Hospital Infection Society (HIS) advises that a plenum-ventilated operating theatre should require a minimum of 15 min before proceeding to the next case after a 'dirty' operation [ 16].
