Formaldehyde

Histomorphometric analysis was performed at the Hard Tissue Laboratory, Faculty of Medicine, University of Coimbra, Portugal. Bone biopsy samples measuring 6 to 8 mm in length were fixed with 10% formaldehyde (Panreac, Barcelona, Spain) solution buffered at pH 7.4 and stored at 4 °C, until histology analysis of non-decalcified hard tissues with the high-precision Exakt® system (Exakt Technologies, OK, USA). The blocks were sectioned longitudinally in 50–100-μm-thick slices, then stained with toluidine blue and examined with a light microscope (Nikon® Eclipse E600, Tokyo, Japan). The new bone formation, the remaining biomaterial, and the marrow space were quantified in percentages using Bioquant® (Image Analysis Corporation, Nashville, TN).

An autovaccine containing 1 × 10⁸ cfu/ml of the C. pseudotuberculosis isolated from the CLA-infected ibexes, and a strain of Archanobacterium haemolyticum previously isolated at the Department of Animal Health at the University of Córdoba, which presents strong immunogenic properties against other corynebacterial species, was elaborated (unpublished data). Briefly, C. pseudotuberculosis and A. haemolyticum isolates were cultivated in brain heart infusion (Oxoid, Spain) at 37°C for 24 h and inactivated with 0.3% formaldehyde (30-40% w/v, Panreac, Spain). The suspension was centrifuged at 720 g for 15 min at 4°C and the pellets re-suspended in an isotonic saline solution until the pattern 0.5 in the McFarland scale was obtained. The vaccination scheme consisted of 5 ml subcutaneous (sc) doses (2 ml for juveniles), with the first dose on December 2011 (t2) followed by revaccination one month later (t3) and subsequent doses every 6 m, on July 2012 and January 2013 (t4 and t5, respectively). Penicillin G benzatine (1.2 million IU, intramuscular; Benzatard®; Syva, León, Spain) was administered to reduce infection progression. Penicillin G has demonstrated high action against Corynebacterium spp. in antibiograms (Personal communication).

Tumors were harvested and fixed in formaldehyde (3.7% to 4% (pH 7); Panreac, Castellar del Vallès, Spain) for 24 h. The relevant parts were embedded in paraffin, and sections were produced for hematoxylin-eosin staining (HE). Immunohistochemistry (IHC) was done as previously described²¹ using the antibody against Id1 (BCH-1/37-2; 1:1500, Biocheck). IHC to study the expression of CD3 (RM9107; 1:300, Neomarkers, Portsmouth, NH, USA), CD8 (#98941; 1:400, Cell Signaling, Danvers, MA, USA), CD4 (#ab183685; 1:1000, Abcam), and FOXP3 (FJK-16s; 1:200, Thermo) was performed by the Morphology Core Facility at CIMA. Slides were scanned with the Aperio Digital Scanner (Leica, Wetzlar, Germany) and analyzed with ImageJ software (NIH, Bethesda, MD, USA).

Fibroblasts were seeded on glass coverslips (Menzel-Gläser, Braunschweig, Germany) and when they reached an appropriate confluence (approximately 80%), they were incubated with FB (0.5 or 1 mg/mL) for 24 h. Cells were then washed with PBS and irradiated with wIRA light. After treatment, cells were fixed with 3.7% formaldehyde (Panreac, Barcelona, Spain) and then permeabilized with 0.01% Triton X-100 in PBS before incubation with specific primary antibodies for matrix metalloproteinase-1 (MMP-1), cathepsin K (CTSK) and fibrillin 2 (FBN2) (Table S1). Afterwards, coverslips were incubated with specific anti-IgG secondary antibodies coupled to AlexaFluor488 or AlexaFluor546 (Table S1). Finally, coverslips were mounted on slides using ProLong^® Gold Antifade Mountant medium containing DAPI (Thermo Fisher Scientific, Waltham, MA, USA).

After osteogenic and adipogenic induction, cells on chamber slides were fixed with 4% paraformaldehyde. For osteogenic differentiation, cells were stained with Alizarin Red, and slides were mounted with DPX mounting medium (Surgipath, Leica Microsistemas S.L., Barcelona, Spain). For adipogenic differentiation, cells were stained with Oil Red O, and slides were mounted with Glycergel aqueous mounting medium. Chondrogenically-differentiated cell aggregates were fixed with 3.7% formaldehyde (Panreac Química S.L.U., Barcelona, Spain), embedded in paraffin (Merck Millipore, Merck KGaA, Darmstadt, Germany) and cut in a microtome.
Micrographs of slides were taken with the Olympus DP70 digital camera coupled to the Olympus BX61 microscope and using the cellSens Dimension software. Stained areas and staining intensity were evaluated quantitatively employing the ImageJ software (National Institutes of Health, Bethesda, MD, USA). To measure staining intensity, optical density (OD) was calculated using the formula OD = log (max intensity/mean intensity), where log is the natural logarithm. An average of optical density and percentage of staining was obtained from four different areas of each sample.

Gantrez^®® AN 119 was supplied by Ashland Inc. (Barcelona, Spain). Mannosamine hydrochloride, zein, mannitol, lysine, tween 20, bromoethylamine-hydrobromide and Bovine Serum Albumin (BSA) were purchased from Sigma-Aldrich (Spain). Acetone was obtained from VWR-Prolabo (Spain) and O- phtalaldehide was provided by Invitrogen (Thermo Fisher, Waltham, Ma. USA). Ethanol, formaldehyde, NaOH and DMSO was supplied by Panreac (Spain). TSB was obtained from bioMérieux (Marcy l’Etoile, France). RPMI was obtained from Gibco-BRL (UK). Coomassie brilliant blue and sample buffer was purchased from Bio-Rad (Spain). All other reagents and chemicals used were of analytical grade.