Sodium metaperiodate

A total of 723 serum samples of hunted Eurasian wild boar from four different German Federal States, namely Rhineland-Palatinate (RP), North-Rhine Westphalia (NRW), Mecklenburg-Western Pomerania (MWP), and Brandenburg (BB), were investigated for antibodies against O. erraticus tick saliva antigen. The samples were obtained within the framework of an ongoing classical swine fever (CSF) surveillance program in the respective Federal States and were kindly provided by the competent regional veterinary laboratories (Rostock, Krefeld, Koblenz, and Frankfurt/Oder). The sampling areas were chosen to reflect the spatial extent of Germany (areas in the North-West, South-West, North-East, and East) and were taken from the sample collection at random.
The samples were transferred to the OIE-Reference Laboratory for ASF—Universidad Complutense de Madrid (UCM) to be tested in ELISA. All serum samples were analyzed against the SGE of O. erraticus and samples giving doubtful results were reanalyzed after deglycosylating the SGE with sodium metaperiodate to eliminate cross-reactivity by glyscosylated epitopes [Oleaga-Pérez et al., 1994]. The test shows an overall specificity of 100 % with experimental sera which drops to about 90 % under field conditions.
Sample results were normalized considering the controls of their plates (two positive controls and one negative control), and the sample to positive ratio (SP ratio, SP) was calculated for each sample. For the samples analyzed repeatedly in different plates, the maximum optical density (worst case scenario) was employed for the calculation of the final results.

Specimens were post-fixed in 1% OsO₄ for 1 h at 4°C and then they were dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections (40–50 nm) of U87MG were cut at ultramicrotome. Similarly to fixing, post-fixing and embedding procedures were validated on pilot studies and they were reported by current literature as optimal conditions for immunogold-based ultrastructural morphometry. We avoided routine method, which consists in avoiding osmium post-fixing and embedding specimens in acrylic resins after fixing with aldehydes only. In fact, such a procedure despite preserving epitopes impairs the preservation of the finest sub-cellular architecture. The method employed here, which combines aldehyde and mild OsO₄ as first and second fixing steps allows minimal epitope covering while preserving cell architecture and providing an optimal “contrast effect” of various cell compartments. This method allows preserving sub-cellular structures with acceptable epitope integrity (Bendayan and Zollinger, 1983 (link); D’Alessandro et al., 2004 (link)).
In fact, osmium enhances the contrast of various cytosolic compartments by marking membranes phospholipids, as clearly confirmed by Swanlund in describing the gold standard of TEM procedures in studying autophagy (Swanlund et al., 2010 (link)). Again, the binding of osmium to cell membranes prevents the formation of membranous artifacts, which may mimic ATG vacuoles.
Post-fixed samples were then embedded using epoxy resin. We used epoxy resin, instead of acrylic resin such as LR White, since it is well-established and commonly used as embedding media for TEM, allowing an optimal ultrastructural resolution.
The post-embedding was carried out collecting ultrathin sections on nickel grids and incubating them in aqueous saturated sodium metaperiodate (NaIO₄) for roughly 30 min at room temperature in order to remove OsO₄ from the samples. After washing with PBS pH 7.4, ultrathin sections were processed for immunocytochemistry. The NaIO₄ is an oxidizing agent which attacks the hydrophobic alkane side-chains of epoxy resin (Bendayan and Zollinger, 1983 (link); Causton, 1984 ) making the sections more hydrophilic and allowing a closer contact between immunogold-conjugated antibodies and the antigens exposed on the surface of each section. The solution enables the detection of specific immunogold placement within a context of subcellular integrity, which allows counting molecules within well-delineated specific cell compartments.

In this experiment the chemicals and reagents have been chosen in such a way that is cost effective and serve the purpose well. Also, the reagents have been chosen according to the purpose, quality, price and availability. Enzymes α-amylase, glucoamylase and glucose isomerase were purchased from Sigma (Switzerland), Tokyo chemical industry (Japan) and Noor enzyme (India) respectively. Corn starch, D-fructose, sodium metaperiodate, H₂SO₄, NaOH, NaCl, HCl and CaCl₂ were purchased from Merck (Germany). Other reagents like sodium metaperiodate and resorcinol were purchased from Merck (India) and Loba Chemical (India) respectively. The cellulosic materials such as sawdust, sugarcane bagasse and rice bran were collected from local markets (Savar, Dhaka, Bangladesh).

Chlorogenic acid (CQA; ≥95%) was obtained from BLDpharm (Shanghai, China). L-Tryptophan (≥98%), sodium metaperiodate (NaIO₄), sodium hydroxide, L-cystein (≥97%), and ascorbic acid (≥99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carmine (E 120, ≥43%) was acquired from Symrise AG (Holzminden, Germany). Hydrochloric acid was purchased from VWR International GmbH (Darmstadt, Germany).

Cellulose was extracted from the date palm waste, sodium hydroxide (NaOH), sodium metaperiodate (NaIO₄), hydroxylamine hydrochloride, 4-nitrophenol, ethylene glycol, 2-aminophenol (OAP), and cinnamaldehyde (CAL) and other solvents were obtained from Sigma Aldrich (St Quentin Fallavie, France) and used as received. Cobalt (II) acetate tetrahydrate was supplied by Fluka (St Quentin Fallavie, France). Acetic acid and sodium borohydride were purchased from VWR CHIMICALS (Rosny-sous-Bois, France).