Ax70trf

Formalin-fixed parenchyma samples were dehydrated in increasing concentrations of ethanol (70°C, 95°C, and 100°C), cleared in Histochoice (Sigma-Aldrich, St. Louis, MO), and embedded in Paraplast (Sigma-Aldrich). Blocks were sectioned serially at a thickness of 10 μm and stained with hematoxylin-eosin.
After qualitative evaluation of the slides, 10 randomly selected digital images of parenchyma were obtained for each heifer. For this, we used a camera (AxioCam HRc; Zeiss, Göttinger, Germany) linked to a light microscope (AX-70 TRF; Olympus Optical, Tokyo, Japan) at 10 × magnification (Figure 1). The resultant images were analyzed with ImageJ 1.48 software (National Institutes of Health, Bethesda, MD). The number of intraparenchymal adipocytes was manually counted in each image, and then the average intraparenchymal adipocyte number was calculated for each animal before statistical analyses. We measured the proportion of mammary epithelial cells in the parenchymal tissue by counting points in the 10 digital images described above. Briefly, a grid of 315 evenly distributed test points was superimposed over the screen image of every digital image. In each animal, the number of test points overlying mammary epithelial cells was counted; with this method, a higher number of points equated to more mammary epithelial cells.

Seeds collected every 15 days starting from anthesis were fixed in FAA (formaldehyde, glacial acetic acid, 50% ethanol, 1:1:18, volume-volume) for 48 h, stored in 70% ethanol, dehydrated in an ethanol series and embedded in 2-hydroxyethyl methacrylate (Historesin, Leica Instruments, Nußloch/Heidelberg, Germany). Cross sections 5-μm thick were obtained with a rotary microtome (model RM2155, Leica Microsystems, Deerfield, IL) and stained with 0.05% toluidine blue, pH 6.5 (O'Brien et al. 1964) . Glass slides were mounted with synthetic resin (Permount, Fisher Scientific, Waltham, MA). Lugol reagent (Johansen 1940) was used for starch detection on sections of samples collected at 120, 135, 150 and 180 DAA. Five samples from each phase were evaluated with a total of 20 cuts per sample. For each cut, five fields were photographed and analyzed.
Image capture, documentation and analysis were performed using a light microscope (model AX70TRF, Olympus Optical, Tokyo, Japan) equipped with a U-photo system and a digital camera (model AxioCam HRc, Carl Zeiss, Jena, Germany). Samples from each collected period were then associated with the respective seed developmental stage.

Leaf discs were collected from the center of the third leaf and fixed in FAA50 (Formaldehyde, acetic acid and ethanol 50%) for 48 h, and then stored in ethanol 70% according to Johansen (1940) (link). Then the plant material was dehydrated in ethanolic series and included in methacrylate (Historesin-Leica), according to the manufacturer's recommendations. For light microscope observation (AX-70 TRF, Olympus Optical, Tokyo, Japan), cross sections 5 µm thick were obtained with an automatic advanced rotary microtome (model RM2155, Leica microsystems Inc., Deerfield, USA), were stained with toluidine blue then photographed using a digital camera (Zeiss AxioCam HRc, Göttinger, Germany). Anatomical features, as leaf thickness and thickness cell layers, were evaluated using Image J (NIH, Bethesda, Ma).