Microm hm 340e

Eyes with ocular adnexa and extraorbital lacrimal glands were surgically excised, fixed in 10% formalin, and embedded in paraffin, and 8-µm sections were cut using a microtome (Microm HM 340E, Thermoscientific Wilmington, DE, USA). Sections were stained with haematoxylin and eosin for evaluation of morphology. Goblet cells in sections were stained with periodic acid-Schiff (PAS) reagent and were examined, photographed and counted with a microscope equipped with a digital camera (Eclipse E400 with a DS-Fi1; Nikon, Inc., Melville, NY, USA) as previously described [75 (link)]. The number of positively stained goblet cells in the superior and inferior conjunctiva was counted, and the length of the basement membrane between the first and last goblet cell was measured. The data are presented as the average number of goblet cells per millimeter per mouse.

Third-instar Ae. aegypti larvae were treated with an LC₅₀ of honokiol (6.5 mg/L) or magnolol (25 mg/L) in all experiments as described above. At 24 h post-treatment, Ae. aegypti larvae untreated and treated with honokiol or magnolol were immediately fixed in Bouin’s fluid^{75 (link)} at 4 °C for 24 h. Larvae were then dehydrated in an ethanol-tetrahydrofuran-xylene series and embedded in Paraplast X-tra (Sigma-Aldrich). The embedded preparations of the larvae were sectioned at a 5 μm thickness using a Microm HM 340E rotary microtome (Thermo Scientific, Walldorf, Germany). The sections were dried at 40 °C overnight, subsequently deparaffinized with CitriSolv (Fisher Scientific, Fair Lawn, NJ, USA), and rehydrated with a series of ethanol in phosphate-buffered saline (PBS) solutions^{30 (link)}. The rehydrated sections were stained in Weigert’s iron hematoxylin for 30 s, followed by Carson’s trichrome staining procedure⁷⁶ . This staining protocol stained the columnar and goblet cells of the midgut blue and red, respectively. These sections were then dehydrated, cleared in xylene, and mounted in EMS Permount (Electron Microscopy Sciences, Hatfield, PA, USA). Images were observed and captured using a DMIL LED microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica MC 170 HD. Observations were taken of 15 larvae under the microscope.

Decellularization protocol optimization was performed on 1 cm-long segments of sciatic nerves. Quantification of remaining myelin, DNA, axonal debris and laminin was performed on 4 cm and 8 cm-long segments of decellularized sciatic nerve tissue. hASC were transplanted in 4 mm-long segments of decellularized common fibular nerves. Dorsal root ganglion (DRG) and spinal cord segment (SCS) were transplanted into 8 mm-long segments of decellularized sciatic nerves. Following decellularization, acellular grafts were either fixed immediately or stored at 4 °C in PBS^−/− overnight for cell transplantation. For proteomics studies, unprocessed tissue was stored at 4 °C in PBS^−/− for 11 days until the decellularization procedure of the experimental condition was completed. For the structural analysis, samples were fixed in 4% PFA at 4 °C for 24 h. The fixed segments were sequentially dehydrated in increasing EtOH concentrations and xylene and embedded in paraffin using a TPC 15 DUO (MEDITE GmbH, Dietikon, Switzerland). The 5 μm-thin cross- and longitudinal sections were made by a rotary microtome (Microm HM 340E; Thermo Fisher Scientific, Waltham, MA, USA).

Isolated tissue was fixed in 4% paraformaldehyde and embedded in paraffin. The 6 μm thickness section was prepared on the Microm HM 340 E rotary microtome (Thermo Scientific, Waltham, MA, USA), and dewaxed in xylene and serial concentrations of ethanol before staining. For hematoxylin-eosin (H&E) and Alcian blue staining, the dewaxed section was stained using the H&E staining kit or Alcian blue staining kit (Servicebio) according to the manufacturer's protocol. For immunohistochemistry, the dewaxed section was boiled in Tris-EDTA antigen retrieval buffer (0.01 M Tris, 1 mM EDTA, 0.05% Tween 20, pH 9.0) for 10 min and incubated with the primary antibody (Supplementary Table S1) at 4 ºC overnight after being blocked with 5% bovine serum albumin (BSA). Then the section was incubated with horseradish peroxidase (HRP)-labeled secondary antibody and stained using the Diaminobenzidine (DAB) Color Development Kit (Servicebio). The section was permanently mounted using the glycerol jelly mounting medium (Servicebio) after dehydrated and clarified in ethanol and xylene. For immunofluorescence, the section was incubated with Alexa Fluor dye-labeled secondary antibody and mounted using the ProLong Diamond Antifade Mountant with DAPI (Thermo Scientific). The images were obtained using the Pannoramic MIDI digital slide scanner system (3DHISTECH, Budapest, Hungary).

Isolated GIT sections were progressively dehydrated in ascending series of alcohol, orientated on transversal section and paraffin embedded. The TMA method used consists in coring automatically the GIT sections in each donor block using a tissue arrayer (TMA Master II^™, 3DHistech LTD) and inserting them in a recipient block (Figs. 2A–2B). From the 80 donor blocks, two recipient blocks of TMA were designed containing 40 samples each (5 sections per animal, from sections 1 to 5 and sections 6 to 10 for block 1 and 2, respectively) (Fig. 2A). Then, three-micrometer sections were obtained from the recipient blocks using a rotary microtome (Microm HM 340E, Thermo Fisher Scientific) (Fig. 2C). Slides were conserved at 4 °C until treatment.

Eyes and ocular adnexa were excised, fixed in 10% formalin, embedded in paraffin, and cut into 5-μm sections using a microtome (Microm HM 340E, Thermo Fisher Scientific). Sections were stained with Periodic Acid Schiff (PAS) reagent to visualize the mucin-containing goblet cells. The goblet cell density was measured in the superior and inferior conjunctiva using NIS-Elements software and expressed as the number of positive cells per millimeter^{22 (link)}. Five to nine eyes per each group were examined. Each image was quantified by two blinded observers.

After fixative perfusion at 1, 2, 7, or 30 days after surgery, pieces of temporal bone tissues were resected and dehydrated for 30 min at each step of an alcohol gradient: once at 30%, 50%, 70%, 85%, and 95%, and twice at 100%. Clearing was performed twice using xylol for 1 h. The specimens were subsequently immersed twice in liquid paraffin (1 h at 42–46 °C), blocked using solid paraffin in a paraffin cast, and incubated at 46–52 °C for 1 day. Deparaffinization was performed for 5 min at each step of an alcohol gradient: once at 30%, 50%, 70%, 85%, and 95% and twice at 100%. Subsequently, the specimens were rinsed twice with distilled water for 5 min. The tissues were cut into 4-μm-thick sections using a Microm HM340E (Thermo Fisher Scientific, Waltham, MA, USA), and cross-sections were stained with hematoxylin–eosin. Histological observation and image acquisition were achieved with an Olympus BX51 microscope (Olympus, Kyoto, Japan). Slices were also stained with Fluoro-Jade C (Fluoro-Jade C Ready-to-Dilute Staining Kit, Biosensis, Thebarton, Australia) to check cellular degeneration according to the manufacturer’s protocol.