Bx60 microscope

Images were captured with an Olympus BX60 microscope, a Leica DM6000 epifluorescent system or a laser confocal microscope (Leica Sp5). For cell counts in sections from wild type (WT) and homozygous brains, we blindly counted the positive cells within equally sized frames (size of frames provided in the figure legends) on coded cross sections of somatosensory cortex in WT and mutant mice (n ≥ 3 per genotype). For BrdU birthdating experiments, frames spanning the entire cortex on cross brain sections were divided into 10 equally-sized bins, the BrdU⁺ cells in every bin were counted and divided by the total number of BrdU⁺ cells in all 10 bins. The size of the counting frames for BrdU/marker co-localization are provided in the figure legends.
Laser confocal microscopy was used to verify co-localization of multiple fluorescent signals. We performed Z sectioning at 0.5-1 μm intervals and optical stacks of at least 10 images were used for analysis, by the Leica Advanced Fluorescence software version 2.3.6. All images were processed with Adobe Photoshop (Version CS2) by overlaying the pictures, adjusting brightness, contrast and size.

Leaf samples were manually excised from the plants, following the rosette formation. Fresh tissue was washed and then cooled to 4° to be sectioned with a razor blade by hand in sections of ~50 μm, to be immediately stained.
Toluidine blue staining was used to differentiate tissue structures based on^{19 (link)}. Following the sectioning, samples were drawn in 1 mL of toluidine blue solution (0.05% w/v in phosphate buffer 1 M at pH 6.8) for 3 min, then washed for 1 min in deionized water and visualized under bright field microscopy (OLYMPUS BX60 microscope and Leica E24HD stereoscope). To identify total carbohydrates on fresh tissue sections, Periodic Acid-Schiff (PAS) and Lugol staining were carried out and visualized as described in^{14 (link)}.
Measurements of agave leaves such as weight, volume and density of excised tissues were registered. Excised leaves were dried at 60° for 10 days (Boekel oven), and the loss of water was monitored every 24 h, until its constant weight. A water displacement method to determine the volume of agave leaves was used in a graduated cylinder before and after the drying process. Water content was experimentally determined (Eq. 1) while density [g/cm³] was calculated as follows: $${\rho }_{\mathrm{leaf}}=\frac{W_{\mathrm{dry}}}{V_{\mathrm{dry}}}$$

Fresh tissue from three plants was washed and then cooled to 4°C to be sectioned using a microtome blade. Alternatively, samples were hand sectioned with a scalpel or a razor blade and used immediately for staining.
The Periodic Acid-Schiff (PAS) stain for total carbohydrates was used for in situ carbohydrate localization. Samples were covered with 500 μL of periodic acid solution (0.5% v/v) and incubated at RT for 5 min and then washed with distilled water for 1 min. Then, 500 μL of Schiff reagent was added to the washed slides and left at RT for 15 min. For starch localization, Lugol dye (500 μL) was added to sample slides and incubated for 15 min. After staining, slides were washed for 1 min before being visualized under bright field and phase contrast microscopy (OLYMPUS BX60 microscope and Leica E24HD stereoscope (Zavala-García et al., 2018 (link)).