Bioclear

For the immunohistochemical studies, tumor tissues fixed in formalin and included in paraffin were used. Paraffin sections (thickness, 3 μm) of the tumor specimens were deparaffinized in Bioclear (06-1782D; Bio-Optica, Milan, Italy) for 30 min, rehydrated in 100%, 90% and then 70% ethanol, and washed with PBS and then PBS containing 0.02% Triton-X 100 (215680010; Acros Organics, Geel, Belgium). The streptavidin–biotin–peroxidase method was used for the detection of synaptophysin (1:200; GA660, Dako, Glostrup, Denmark). The antigenic was recuperated with sodium citrate (pH 6.0) for 30 min. The endogenous peroxidase activity was blocked with 3% hydrogen peroxidase (H2O2). Incubation with primary antibodies was performed overnight at 4 °C. Incubation with secondary antibodies was performed for 30 min at room temperature. Then, antigen–antibody binding was visualized.

Genomic DNA was isolated from biopsy tissue sections using a standard protocol. In particular, paraffin was removed from formalin-fixed paraffin-embedded (FFPE) samples with Bio-Clear (Bio-optica, Milan, Italy), and DNA was purified using the QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, CA, USA). DNA quantitation and quality assessment were carried out with both a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA fragmentation status was evaluated with an Agilent 2200 TapeStation system using Genomic DNA ScreenTape assay (Agilent Technologies, Santa Clara, CA, USA) able to produce a DNA integrity number (DIN). Quantitative measurements of EGFR mutations were performed using the Therascreen™ EGFR Pyro Kit (Qiagen), for the detection of mutations in codon 719 (exon 18), in codons 768 and 790 (exon 20), and in codons 858 to 861 (exon 21), along with deletions and complex mutations in exon 19 of the human EGFR gene on genomic DNA. Each pyrosequencing assay was performed on the PyroMark Q24 system (Qiagen), following the manufacturer’s instructions.

Air-dried sections were blocked with 5% normal horse serum in PBS, to avoid non-specific binding of the antibodies, and incubated with primary antibodies in a humidified chamber, at room temperature, for 2 h. Specimens were washed three times with PBS and then incubated 1 h with an anti-mouse biotinylated secondary antibody raised in horse, washed, and incubated with VECTASTAIN ABC Reagent (Vector Laboratories, Burlingame, CA USA) for 30 min. After washing, the sections were incubated in Diamino Benzidine solution (SigmaFastDAB; Sigma Aldrich, Milano, Italy), until development of staining. Sections were dipped in water, dehydrated with 95% and absolute ethanol alcohol, followed by clarification in BioClear (BioOptica, Milano, Italy), and mounted with a resinous medium. Images were acquired with a Leica Orthoplan microscope (Leica, Wetzlar, Germany) equipped with a Leica DC-100 digital camera (Leica, Wetzlar, Germany).

Muscles samples were fixed in 4% paraformaldehyde phosphate-buffered saline (PBS) for 3 h, washed in PBS and embedded in paraffin after dehydration with ascending ethanol passages (50, 70, 80, 95, 100%) followed by diaphanization in Bioclear (Bio-Optica, Milano, Italy). Gastrocnemii were sectioned (7 µm thick) using an RM2135 microtome (Leica Microsystems) and placed on slides. They were then deparaffinized and rehydrated with decreasing ethanol passages.
Immunohistochemistry staining was performed using IHC Select^® HRP/DAB (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Briefly, slides were treated with 0.1% trypsin solution to recover tissue antigenicity. Then, 3% hydrogen peroxide solution was used to block endogenous peroxidase activity. After an incubation of 5 min in blocking reagent, slides were incubated overnight with primary antibodies (Supplementary Table S1) at 4 °C. The next day, the secondary antibody included in the kit was added to the slides for 10 min, followed by a first incubation with streptavidin HRP (10 min) and then a second incubation with the chromogen reagent (8 min).
The sections were dehydrated with ascending ethanol passages and mounted in dibutylphthalate polystyrene xylene (DPX), (BDH) [28 (link)].

After fixation and paraffin embedding, we cut the scaffolds (3D fibroblasts) into 5–10 μm thick slices using a microtome (Leika, Wetzlar, Germany) and stuck them onto glass slides coated with polylysine (Superfrost Plus, ThermoFisher Scientific, Waltham, MA, USA). The sections were cleared in xylene substitute (Bioclear, Bio-Optica, Milan, Italy) and dehydrated by immersion in ethanol (Sigma-Aldrich, St. Louis, MO, USA; from 100% to 50% serial dilutions).
We embedded the fixed hydrogels (3D HUVECs) in an Optimal Cutting Temperature (OCT) compound, froze them in liquid nitrogen, and sectioned them at 10 μm thickness using a cryostat (Leica Biosystems, Weitzlar, Germany) at −25 °C. We stuck the sections onto glass slides and stained them: (i) with H&E (Bio-Optica, Milan, Italy) to evaluate tissue morphology and structure and (ii) with GTR to evidence scaffold remodeling and new ECM deposition. We analyzed the 3D growth/organization of the cells both in static and dynamic environments by light microscopy (Leica FC7000 T, Leica Biosystems, Weitzlar, Germany).

After decapitation, the brain was rapidly removed, and processed to prepare cryostat sections, essentially as previously described [76 (link)]. Briefly, the left/right half of the brain was embedded in killik (Bio-Optica, Milan, Italy), frozen in liquid nitrogen and sectioned using a cryostat (Leica CM1900UV, Wetzlar, Germany). 10–15 µm-thick coronal sections were collected on poly(L)-lysine-coated slides and stored at −20 °C until further processing. Selected sections were thawed at RT, stained with cresyl violet (Bio-Optica, Milan, Italy), differentiated in two changes of 95% ethanol, quickly dehydrated with absolute ethanol, cleared with Bio-clear (Bio-Optica, Milan, Italy) and permanently mounted with Eukitt (Bio-Optica, Milan, Italy). Whole-slide images were acquired using the Manual WSI software (Microvisioneer, Esslingen am Neckar, Germany) and a BX60 microscope (Olympus, Hamburg, Germany) equipped with a color camera (Basler, Ahrensburg, Germany).

Total RNA was extracted from paraffin-embedded samples of liver and kidney (5 samples/treatment group) using the RNeasy FFPE Kit (Cat. No./ID: 73,504; QIAGEN, Düsseldorf, Germany) according to the manufacturer’s instructions. Briefly, paraffin was removed from 10 μm thick sections by Bio Clear (Bio-Optica, Milano, Italy), then proteinase K tissue digestion was performed (10 μm; 56 °C for 15 min). To reverse formalin crosslinking, samples were incubated at 80 °C for 15 min. Genomic DNA was removed by DNase digestion, RNA was quantified by Nabi Nano Spectrophotometer (MicroDigital Co., Ltd., Seoul, Republic of Korea) and ran on 1% agarose to evaluate its integrity. Despite poor yield, all the samples met the quality criteria (A260/A280 ≥ 1.8) to proceed with real-time PCR analysis. cDNA synthesis and qRT-PCR were performed [25 ]. The list of primers is provided in Supplementary Materials (Table S1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control gene for RNA normalization. The relative amount of each substrate was calculated by the 2−∆∆Ct method.