Sigmafast

Antibody production was quantified from the peripheral blood of mice obtained at 0, 15, 30, and 45 days after immunization using the indirect ELISA method [25 (link)]. For this, 96-well plates (Nunc Maxisorp, Thermo Fisher Scientific, MA, USA) were coated with 5 μg of recombinant proteins per milliliter of carbonate-bicarbonate buffer (50 mM, pH 9.6) and incubated for 16 h at 4°C in a humidity chamber. Plates were washed with TPBS buffer (PBS plus 0.05% Tween 20) and blocked with 0.8% gelatin for 1 h at 37°C in order to avoid nonspecific binding. To each well, 100 μl of serum was added in serial dilutions using TPBS plus 0.2% gelatin, starting at 1 : 200 dilutions. Samples were incubated for 3 h at 37°C. After this time, rabbit anti-mouse IgM, IgG, IgG1, or IgG2a secondary antibodies conjugated with horseradish peroxidase (Serotec, Oxford, UK) diluted 1 : 1000 were added and incubated for 45 min at 37°C. The reaction was revealed with 100 μl SigmaFast (Sigma-Aldrich, St. Louis, MO, USA) OPD. The final reaction was stopped with 50 μl of sulfuric acid 2 N and read at 490 nm using a VictorX3 microplate reader (PerkinElmer, USA). Results were expressed as mean ± standard deviation (SD) of the inverse from the last dilution reached before the cut-off. All experiments were done in triplicate.

The hearts were homogenized in lysis buffer containing (mmol/l) 150 NaCl, 50 Tris-HCl, 5 EDTA.2Na, and 1 MgCl₂ plus protease inhibitor (Sigma Fast; Sigma, USA). The protein concentration was determined by the Lowry method, [22] (link) and bovine serum albumin (BSA) was used as the standard. Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes incubated with mouse monoclonal antibodies for catalase (CAT; 1∶2000; Sigma, USA), rabbit polyclonal antibodies for superoxide dismutase (SOD-2; 1∶1000; Sigma, USA) and Gp91phox (1∶1000; BD, New Jersey, EUA) and rabbit polyclonal antibodies for AT₁ (1∶500; Santa Cruz Biotechnology, CA, USA) and GAPDH (1∶1000; Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated with either an alkaline phosphatase conjugated anti-mouse IgG (1∶3000, Abcam Inc., Cambridge, MA, USA) or an anti-rabbit antibody (1∶7000; Santa Cruz Biotechnology, CA, USA). The bands were visualized using a NBT/BCIP system (Invitrogen Corporation, CA, USA) and quantified using ImageJ software (National Institute of Health, NIH). The results were calculated using the ratio of the density of specific proteins to the corresponding GAPDH.

1.25 µg of Lactate dehydrogenase from rabbit muscle (Sigma-Aldrich®, Sigma, USA) was denatured with 1 molar guanidine hydrochloride (GdnHCl) during 10 min at 25 °C. After that, 100 mM of NaH₂PO₄ pH 7 buffer was added, the final volume mix was 500 µl. The mix was incubated at 25 °C for 90 min with 287 nM chaperones: BSA (negative control), OppAWT, OppA mutants and HtrA (as positive control). Again, The SIGMAFAST™ (Sigma, USA) Protease Inhibitor was added according to the protocol of the commercial kit. Lactate dehydrogenase (71.43 nM) activity assays were performed at 25 °C according to Sigma-Aldrich® (Sigma, USA) protocol. β-NADH was measured by spectrophotometry at 340 nm every 10 s for 5 min. LDH units in each assay were calculated according to LDH activity assay protocol from Sigma-Aldrich® (Sigma, USA).
The molar ratio beween LDH and chaperone-like proteins (OppA, BSA and HtrA) was set to the same proportion as before, 1 to 4, while GdnHCl was applied at a 20 mM concentration because at this concentration level the LDH activity remains unaltered, all of which had been empirically determined prior to our bioassays.

Animals in group B (n = 4) were used for IHC analysis on day 44 after 14 days of treatment. Animals were euthanised with an overdose of sodium pentobarbital (Mebunat vet 10 mg/100 g, intraperitoneal injection, Orion Pharma, Turku, Finland). The blood was then removed via cardiac puncture and perfused first with heparinised saline with paraformaldehyde-lysine-periodate light fixative containing 0.1% glutaraldehyde. The brains were cryoprotected in 30% (w/v) sucrose solution. IHC staining was performed using an antibody (Ab) against the ionised calcium-binding adapter molecule 1 (Iba-1, Wako 019-19741, Rabbit-anti-rat, Wako Chemicals GmbH, Neuss, Germany) to detect microglia. The sections were incubated with anti-Iba-1 primary Ab overnight at 4 °C. After this, the sections were incubated with anti-rabbit IgG (Invitrogen, Camarillo, CA, USA) for 1 h at RT. Finally, they were incubated with avidin-biotin-peroxidase complex (Vectastain Elite kits, Vector Laboratories, Burlingame, CA, USA). The sections were stained with 3,3′-diaminobenzidine (SIGMAFAST, Sigma-Aldrich, Saint Louis, MO, USA) and counterstained with cresyl violet to stain the nuclei.

Mice colon tissues fixed in 10% buffered formalin and embedded in paraffin were stained for NOX2. The tissue sections were deparaffinized with Xylene (catalog no. 9800-1, Caledon) and rehydrated sequentially in graded concentrations of ethanol. After heat-induced epitope retrieval, tissues were blocked with 3% bovine serum albumin and incubated with a rabbit monoclonal NOX2/gp91phox antibody (1:200; catalog no. ab129068, Abcam) for 1 h at room temperature. The sections were washed with phosphate-buffered saline/0.5% Tween 20 and then incubated with EnVision (horseradish peroxidase-coupled anti-rabbit secondary reagent; DakoCytomation, catalog no. K4003, Dako) for 30 min. The development of the sections was performed using a 3,3′-diaminobenzidine solution (SIGMAFAST, catalog no. 079K8208, Sigma-Aldrich) and counterstained with Mayer’s hematoxylin solution (catalog no. MHS1, Sigma-Aldrich). The sections were finally visualized using a Nikon Eclipse 80i microscope (Nikon Instruments Inc. Melville, NY, USA).