Jb 4 embedding kit

HREM analysis was done to four plants from each genotype. Tissues were fixed overnight in 4% paraformaldehyde (PFA) (v/v), rinsed in PBS buffer and dehydrated through an ethanol series up to 95% ethanol. Fixed and dehydrated tissues were infiltrated with catalyzed monomer A of the JB-4 embedding kit (Electron Microscopy Sciences) containing eosin and acridin orange dyes as previously described (Weninger and Mohun, 2007 (link); Pokhrel et al., 2017 (link)). Samples were incubated for 7 days on a revolving shaker at room temperature in the dark. Samples were positioned and mounted in plastic molds and sectioned using the HREM system (Indigo Scientific, Baldock, United Kingdom). Images of >800 sections (2.5 μm thick) were captured, stacked, and processed using Fiji software (Schindelin et al., 2012 (link)) and 3D reconstruction was performed using Amira software (FEI, Hillsboro, OR, United States).

Mineral deposition was evaluated by intraperitoneal injections of calcein (Sigma # C0875; 2.5 mg/kg body weight) and alizarin complexone (Sigma # a3882; 7.5 mg/kg) into pregnant females and, postnatally, into cubs. Two mice were used for each injection regime. Prenatally harvested limbs were fixated overnight in 4% PFA/PBS, dehydrated to 100% ethanol, embedded in paraffin and sectioned at a thickness of 7 μm. Postnatally harvested limbs were fixated 24 h in 4% PFA/PBS and gradually dehydrated from 70% ethanol to 100% ethanol twice for 48 h each time. Then, samples were infiltrated and embedded in JB-4 Embedding Kit (Electron Microscopy Science #14270–00) and sectioned longitudinally at a thickness of 7 μm. Fluorescence was visualized by confocal microscopy.
Confocal imaging was performed using a Zeiss LSM 510 upright confocal microscope (Carl Zeiss, Jena, Germany) with an EC Plan-Neofluar 10x/0.3 objective, NA 1.0. Calcein fluorochrome was excited with a 488 nm argon laser and alizarin with 561 nm argon laser. Following imaging, all images of the same section were stitched using Microsoft Image Composite Editor (version 1.4.4.0). Contrast was increased by using the “auto contrast” tool of Google Picasa (version 3.9.137) and Matlab’s “imadjust” function.

Histologic analysis was done to 10 plants from each genotype. Tissues were fixed overnight in 4% paraformaldehyde (PFA) (v/v), rinsed in PBS buffer and dehydrated through an ethanol series up to 95% ethanol. Fixed and dehydrated issues were infiltrated with catalyzed monomer A of the JB-4 embedding kit (Electron Microscopy Sciences) and embedded into JB-4 plastic resin under an oxygen-free environment. Blocks were serially sectioned (interval 4 mm) on a rotary microtome (Leica RM2255) with TC-65 disposable blades (Leica). Sections were stained with 0.1% (w/v) toluidine blue prior to examination and photographed on an Olympus BX53 digital microscope equipped with an Olympus DP73 digital camera using bright field.