Paraplast x tra

Adult mice were perfused with 4% parafolmaldehyde (PFA) in PBS or tissue pieces and embryos were fixed overnight with 4% PFA at 4°C. Fixed tissues were dehydrated in graded alcohol series, embedded in paraffin (Paraplast X-tra, Sigma-Aldrich), sectioned into 8 μm thick sections and stained with Haematoxylin-Eosin (H and E) using standard protocols. For immunostainings, sections were hydrated with inverse graded alcohol series, unmasked by heating in 10 mM citrate buffer (pH 6) for 10 min, blocked for 1 hr with 3% BSA at RT and incubated overnight with the primary antibody, washed, incubated with secondary antibodies for 1 hr at room temperature, washed, DAPI stained and mounted on glass slides with elvanol.

Leaf tissue was harvested 4 hrs after dawn and immediately infiltrated in ethanol∶acetic acid (3∶1). The tissue was processed through a series of dehydration and then replaced by Paraplast Xtra (Sigma). Leaves embedded in wax were sectioned transversely using 8 µm thin sections. Sections were floated in EtOH on MembraneSlide 1.0 PEN (Zeiss) and dried. For laser capture microdissection (LCM), slides were deparaffinised using Histo-clear for 2 min and air dried. LCM was performed using Arcturus XT (Life Technologies) and mesophyll and bundle-sheath were captured using adhesive caps (Life Technologies) following manufacturer instructions. Subsequently RNA was purified using Picopure RNA extraction kit (Life Technologies) and subjected to on-column DNAse treatment (Qiagen) and amplified using Nugen RNA Ovation V2 kit (Nugen) according to the manufacturer's instructions. RNA quality and quantities were checked at every stage using a picoRNA chip on Bioanalyzer 2100 (Agilent). Amplified cDNA libraries were using the Illumina standard protocol and then multiplexed on HiSeq to generate 2 Gb of 100 bp pair ended reads for each library (in triplicate for each cell type).

Subcutaneous adipose tissue samples (50–100 mg) were obtained after carefully excision with a scalpel blade and fixed in 4% paraformaldehyde (w/v) in pH 4.7 Phosphate buffer, followed by dehydration in absolute ethanol, diaphanization in xylene and embedding in paraffin (Paraplast X-TRA, SIGMA-ALDRICH). The 5 μm sections were mounted onto slides (Starfrost® Knittel Glass). Deparaffinized and hydrated sections were stained with hematoxylin and counterstained with eosin (n = 05 for all study groups) and photographed under light microscopy (Olympus).

Adult chinchilla-breed virgin female rabbits (Oryctolagus cuniculus) of 8–9 months were housed under controlled temperature (20 ± 2 °C) conditions and a 16:8 h light: dark cycle. Animals were provided pellet food (120 g/day) and water ad libitum. Rabbits were randomly assigned to the following experimental groups: control (control; n = 6), moderate oral (drinking water) dose of I₂ (0.2 mg/kg; M-I₂; n = 3), a high oral dose of I₂ (2.0 mg/kg; H-I₂; n = 4); oral dose of methimazole (10 mg/kg; MMI; n = 6), MMI + M-I₂ (n = 6), and MMI + H-I₂ (n = 6). The body weight of the females was measured before and at the end of treatments. After four weeks of treatment, the rabbits were anesthetized with sodium pentobarbital (90 mg/kg, i.p.) and euthanized with an overdose of the same anesthetic. The Ethics Committee at Universidad Autónoma de Tlaxcala approved this experimental design following the guidelines of Mexican Law for the Production, Care, and Use of Laboratory Animals. Immediately after death, the left lobe of the pancreas was collected, histologically processed, embedded in Paraplast X-TRA (Sigma-Aldrich, St Louis, MO, USA), and longitudinally cut at a thickness of 5 μm using a microtome (Thermo Scientific, Model Finesse 325, Waltham, MA, USA). The right lobe of the pancreas was frozen at −80 °C for biochemical measures.

The fixed tissue samples were washed in PBS and dehydrated in a graded series of alcohol before being embedded in Paraplast X-tra® (Sigma). Tissue sections (5 µm) were cut from the wax blocks before staining with haematoxylin and eosin. Images were acquired using a Hamamatsu Nanozoomer-HT-2.0 Digital pathology System (NDP).

Freshly harvested seeds of 4, 5, 7, 9, and 11 DAP were dehusked, fixed, and dried in ethanol series (as mentioned in the previous section). Ethanol was gradually replaced with xylene by keeping the tissues in increasing concentration of xylene and decreasing concentration of ethanol in the percentages of 25:75, 50:50, and 75:25. Next, two rounds of treatment with 100% xylene were given to the seeds at room temperature for 1 h on the rocker, followed by infiltration with paraplast X-TRA (Sigma-Aldrich; USA) by adding molten paraplast every 2–3 h at 60 °C for 3 days. The wax-infiltrated tissues were then embedded into molds (Yorko®; India), and 10-μm sections were cut for 4, 5, and 7 DAP and 15-μm sections were cut for 9 and 11 DAP using rotary microtome (Leica Biosystems, Germany). Paraplast was removed from the sections by immersing the slides in HistoChoice® clearing agent (Sigma-Aldrich; USA) for 1 h. Sections were stained with 0.01% toluidine blue-O for 10 min or 0.1% Coomassie brilliant blue/CBB [0.25% CBB in 50% methanol and 10% acetic acid] for 20 min or 2% I2/KI solution (2 g KI and 0.2 g Iodine in 100 ml MQ) for 2–5 min. The sections were mounted using D.P.X. mountant (Himedia®, India) and were visualized under light microscope (Eclipse 80i, Nikon; Japan) at × 20 magnification.