Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Inferring climate from angiosperm leaf venation networks (2014), New Phytologist. Implementations in other papers (listed below) may differ.
Leaf sample collection
At each site, sampled at least one leaf from at least one individual of every observed species. For tropical sites, chose a random subset of fifty leaves, each from a different individual for venation analysis. For temperate sites, sampled 9 ± 1 SD mature undamaged leaves from individuals of each species.
Quantities: 187 angiosperm species total, 17 sites, 1048 leaves measured for vein density, 225 tropical leaves and 529 temperate leaves in final datasetDuration: Not specifiedConditions: Field collections along elevation gradients during growing seasons
Equipment: GPS units for coordinates, elevation measurement tools
Leaf preservation and clearing
Leaves were pressed flat and dried at 60°C for at least 3 days. Cleared each leaf to expose its venation using established protocols. Selected a region of the lamina that did not include any primary veins. Immersed leaf sample in solution of 5% w/w sodium hydroxide and heated to temperature of 50°C for up to 7 days until leaf became transparent.
Quantities: Each leaf processed individually, heated for up to 7 daysDuration: 3 days drying, up to 7 days clearing processConditions: 60°C drying temperature, 50°C clearing temperature
Equipment: drying oven, heating equipment, sodium hydroxide solution
Chemical processing for microscopy
Rinsed leaf in water and transferred to 2.5% w/v sodium hypochlorite solution for 5 min, then to staining solution of 0.1% w/v safranin : ethanol for 30 min. Transferred leaf to destaining solution of 100% ethanol for 1 h before transferring to 50% v/v ethanol : toluene for 30 s and then to 100% toluene. Mounted each leaf on glass slide using toluene-based Permount medium.