Functional analysis of a putative membrane-bound endo-β-1,4-glucanase from Panicum virgatum

Samples were collected from switchgrass transformed with an overexpression of the Egase. Seven parts from each plant were collected in triplicate including the first three nodes, the leaf tip, the middle of the leaf, the leaf base, and the stem below the first node. These samples were then placed in Formalin-Acetic Acid-Alcohol. The samples were then dehydrated with 95% ethanol for two days. After dehydration, the samples were infiltrated with JB-4 (catalyzed Monomer A) . The first step in this infiltration was to use a solution containing one quarter JB-4 in three-quarters 95% ethanol for five days. Next, a 1:1 ratio of JB-4 to 95% ethanol was infiltrated for two days. Then, three-quarters JB-4 and one-quarter 95% ethanol was infiltrated for two days. Finally, the samples were placed in 100% JB-4 for two days. The samples were then removed and placed in a plastic molding tray. Monomer A was combined with Monomer B to initiate the hardening reaction. The molds were then placed under nitrogen gas and hardened. The samples were then cut using a glass blade on a microtome. The plastic samples were then sectioned in 5 micron sections and placed on slides. The samples were stained with Pontamine Fast Scarlet 4B, then viewed using the 40x objective under identical epifluorescent parameters.