Methyl salicylate

Mammary whole mounts were prepared using fixed left inguinal mammary glands, stained with carmine alum (71 ), and stored at room temperature in methyl salicylate (Sigma-Aldrich, St. Louis, MO). Imaging of each gland immersed in methyl salicylate was performed with a Nikon SMZ 800 dissecting microscope with an attached Olympus DP70 camera and electronic images were captured using DP control software (Olympus Optical Co. LTD. 2002). Measurements were calibrated with a metric ruler, and the parameters were quantified using Image J software in a blinded manner. Elongation of the ducts into the mammary fat pad and the length of the mammary fat pad were determined by measurement from the center of the lymph node to the farthest terminal duct and to the leading edge of the gland, respectively. Tertiary branching was determined by quantifying the number of tertiary branches per mm² in three separate distal ductal areas of each gland to determine the mean number of branches per mm² per mouse.

For embryological processes observation, 23 flowers (KAM, KON, PIL, RUR) were cleared using methyl salicylate method, and divided into two groups. I. Flowers at preanthesis and anthesis stages were cleared in a mixture of 100% ethanol and methyl salicylate (Sigma-Aldrich) in proportions 3:1; 1:1; 1:3, 0:3 two hours each change. II. At postanthesis stages prior to clearing, pre-softening of the tissues using Schiff reagent was applied. Cleared pistils and/or anthers were transferred onto Ray chambers in a drop of methyl salicylate and observed under Nikon Eclipse Ni light microscope with Nomarski differential interference contrast (DIC) optics, equipped with camera Nikon DS-Filc and NIS-Elements BR Viewer imaging software ver. 4.10 (^{57 (link)}, slightly modified). For the study, 10 randomly selected ovules from each flower were analyzed.