Paraplast plus

Animal were euthanised with 10% CO₂ for five minutes at each time point. They were confirmed dead when they stopped breathing, did not have a heartbeat, and did not respond to a firm toe pinch. The animals were shaved, a dorsal midline incision was made using a scalpel, and the adjacent fascia was released via gentle dissection to find the explanted tissue. The dissected tissues were transferred into a bowl filled with 1xPBS before being fixed in the 10% neutral buffered formalin.
The skins with implanted scaffolds, kidneys, and livers of the euthanised animals were fixed in 10% (v/v) neutral buffered formalin overnight and processed and processed in an automated Excelsior™ AS Tissue Processor (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h. Tissues were embedded in paraffin (Paraplast^® Plus, Leica Biosystems, Richmond, IL, USA) and cut into 3 to 5 μm sections with a rotary microtome Histo-Tek SRM II (Sakura Finetek, Torrance, CA, USA). The sections were transferred into a water bath at 37 °C and then mounted on frosted-end slides (HmbG GmbH, Hamburg, Germany) for basic staining.

Mites were fixed in modified Bouin-Dubosque fluid (Smrz, 1989 (link)) for 3 days and then transferred to paraffin. The fixation fluid was replaced by 100% isopropyl alcohol for 12 h (two times), isopropyl alcohol/methyl benzoate (1/1 v/v) for 12 h (two times), methyl benzoate for 12 h (two times), benzene for 2 h, benzene/paraffin (1/1 v/v) for 12 h at 48°C, and paraffin for 12 h (two times) at 56°C (Hubert et al., 1999 ). Mites were transferred from containers to Peel-A-Way^® embedding molds (Polysciences, Eppelheim, Germany) and embedded in Paraplast Plus^® (Cat No. 39602004, Leica Biosystems, Nussloch, Germany) at 56°C. Paraffin blocks were sectioned to 4–6 μm sections on Microm HM 200 ErgoStar Microtome (Carl Zeiss, Jena, Germany).

Inflorescence stem samples were obtained from 3‐week‐old plants from both EV control line and VvCEB1_opt‐overexpressing lines. The samples were fixed overnight at 4 °C in FAA fixative (10% v/v formalin, 5% v/v acetic acid and 50% v/v ethanol) with 0.03% v/v Tween 20. The fixed samples were paraffin‐embedded (Paraplast Plus^®; Leica Biosystems Inc.) after being dehydrated in a series of increasing concentration of ethanol (from 10% to 100% in 10% increments followed by 25%, 50% and 100% tert‐butyl alcohol (TBA) series. The embedded samples were cut into 10‐μm sections using Leica RM 2145 microtome (Leica Biosystems Inc.). The sections were stained using 0.25% w/v toluidine blue in 1× phosphate‐buffered saline (PBS) buffer (137 mm NaCl, 2.7 mm KCl,10 mm Na₂HPO₄, 2 mm KH₂PO₄) after being rehydrated in a series of decreasing concentration of ethanol (from 100% to 10% in 10% decrements), followed by ddH₂O and 1× phosphate‐buffered saline (PBS). Bright‐field images of the stained sections were captured using Keyence BZ‐X710 microscope (Keyence Corporation of America, Itasca, IL).

Mutant and WT fish, heterozygote, and WT mice ovaries were fixed for 24/48 h in Bouin’s solution at room temperature. Samples were dehydrated through a graded ethanol series, infiltrated with xylene, and embedded in Paraplast plus (Leica Biosystem, 39,602,004). Samples were serially cut into 7–8 μm sections on an LKB microtome. After rehydration, sections were stained with hematoxylin and eosin and mounted with Eukitt (BioOptica, 09–00100) for microscopy examination.

Immunolocalization studies were performed using an indirect immunoperoxidase technique. Briefly, the renal tissue samples, 3–4 mm thick, were fixed by immersion in Bouin’s solution for 18–24 h at 21–24°C. Then the samples were dehydrated, embedded in Paraplast Plus, sectioned at 5–7 μm thickness with a rotatory microtome (Leica Biosystems, Germany), and mounted on glass slides. Kidney sections were dewaxed, hydrated and washed in Tris–phosphate buffer, pH 7.6 and incubated overnight with anti-COX-2 at 1:500 (SC-1747, Santa Cruz, CA), at room temperature. This was followed by incubation with the corresponding secondary antibody and with the peroxidase-antiperoxidase complex (MP Biomedicals, Santa Ana, CA). Peroxidase activity was detected by incubation of the sections with 0.1% (wt/vol) 3,3′-diaminobenzidine and 0.03% (vol/vol) hydrogen peroxide. Kidney sections were analyzed as previously described (Villanueva et al., 2008 (link)) using Nikon Eclipse E600 microscopy and Nikon DS-Ri1 camera (Nikon Corp., Tokyo, Japan). Four representative fields for each section were used and an average of six rats quantified. Images were quantified by Simple PCI 6.0 software (Hamamatsu Corporation, Sewickley, PA) and average values of stained areas were expressed as fold change of controls.

The brain samples were flushed and fixed in 10% neutral buffered formalin for 72 h. Then, samples were processed in different grades of ethanol, cleared with xylene, and infiltrated with Paraplast Plus tissue-embedding media (Leica Biosystems, Germany). Using a rotatory microtome, we cut 4 µm thick serial sagittal brain sections and mounted them on glass slides to examine the hippocampal subregions. We then stained the tissue sections with hematoxylin and eosin for pathological examination. We stained another set of tissue sections with toluidine blue to quantify intact neurons and examined them using an optical microscope. We used standard sample-fixation and staining procedures and protocols described by Culling [40 (link)]