Peroxidase conjugated goat anti rabbit igg

Confluent rat C6 glioma cells resuspended in 250 mM HEPES buffer, pH 7.7 with 1 mM EDTA and 0.1 mM neocuproine (HEN buffer) and homogenized. Total protein concentration of the lysate was adjusted to 1 mg/ml. Obtained cell lysates were treated using BST as previously described [37 (link), 44 (link)]. Supernatants from each BST step were collected, proteins were separated using 15% SDS–PAGE and transferred onto PVDF membrane (0.22 μm). After blocking with non-fat dried milk, the PVDF membrane was incubated with goat anti-S100B polyclonal antibody (1:1000 dilution, Santa Cruz Biotechnology) for 1 hour and afterwards for 1 hour with peroxidase-conjugated rabbit anti-goat IgG (1:10000 dilution, Sigma-Aldrich). The peroxidase activity was visualized using the Amersham ECL Western Blotting Detection Reagent (GE Healthcare) with subsequent exposure to X-ray film.

Mice were bled at the time of treatment and at 10 days post treatment, and sera stored at −70°C until use. The specific serum IgG isotype antibody (Ab) response was measured by conventional enzyme-linked immunosorbent assay (ELISA) [14] (link). Briefly, wells of ELISA plates (Nunc, Roskilde, Denmark) were coated with FT (25 µg ml⁻¹) and incubated overnight at 4°C. After washing three times with buffer (20 mM PBS, pH 7.2 containing 0.05% Tween 20), the wells were blocked with 1% BSA for 2–3 h at room temperature. The plate was washed and mice sera at 1,000-fold dilution was added, followed by washing and incubation with isotype-specific goat anti-mouse IgG1 and IgG2a antibody (Sigma Aldrich) at 4°C overnight. The wells were then washed and incubated at 4°C overnight with peroxidase-conjugated rabbit anti-goat IgG (Sigma Aldrich). The wells were washed and incubated with substrate solution (o-phenylenediamine dihydrochloride, 0.8 mg ml⁻¹ in 0.02 M phosphate-citrate buffer, pH 5.0, containing 0.04% H₂O₂) for 30 min, and the absorbance read on an ELISA plate reader at 490 nm.

The Leishmania-specific serum IgG subclasses were measured through enzyme-linked immunosorbent assay (ELISA). In brief, FT (0.25 μg/well) was seeded in the wells of ELISA plates (Nunc, Roskilde, Denmark) for 1 h at 37°C. After three washes, blocking was done with 1% BSA for 2 h at RT followed by addition of 1,000-fold diluted mice sera. Post-washing, IgG1 and IgG2a isotype-specific goat anti-mouse secondary antibodies (Sigma Aldrich) were added and the plates incubated at 37°C for 1 h. After washing, incubation with peroxidase-conjugated rabbit anti-goat IgG as the tertiary antibody (Sigma Aldrich) was done at 37°C for 1 h. Post-washing, OPD was added and the absorbance taken on an ELISA plate reader at 490 nm [41 (link)].

At 3 days after surgery, mouse hippocampal Cornu Ammonis 3 (CA3)/DG regions were collected, and lysed with ice‐cold homogenization buffer containing 2% protease inhibitor cocktail (Thermo Scientific). The samples containing equal amounts of protein were separated on 10% sodium dodecyl sulfate‐polyacrylamide gels (SDS‐PAGE), and transferred to PVDF membranes. After blocking with 5% nonfat milk and 0.1% Tween 20 in Tris‐buffered saline (TBS) at room temperature for 2 h, the membranes were incubated at 4℃ overnight with rabbit anti‐pERK1/2 (1:2500, Cell Signaling), rabbit anti‐ERK1/2 (1:2000, Cell Signaling), rabbit anti‐pAkt (1:2000, Cell Signaling), rabbit anti‐Akt (1:1000, Cell Signaling), and mouse anti‐β‐actin (1:10,000, Sigma). After washing, the membranes were incubated with peroxidase‐conjugated goat anti‐rabbit IgG (1:10,000, Sigma) or goat anti‐mouse IgG (1:10,000, Sigma) for 1 h at room temperature. The target protein signal was detected using a chemiluminescence detection kit (Pierce™ ECL western blotting Substrate, Thermo Scientific) and visualized on an electrophoresis image analyzer (Tianneng). β‐Actin was employed as the loading control. The band intensity was analyzed using Image J software. Quantification of densitometric values was calculated as follow: [(phospho‐protein/β‐actin)]/[total‐protein/β‐actin)] and expressed as ratio relative to the sham group.

rSsEno IgG, IgG1, IgG2a and IgG3 antibody titration was conducted as described by Portuondo et al.^{19 (link)} with some modifications. Briefly, a 96-well ELISA plate (Costar) was coated with 5 μg rSsEno/mL in PBS and incubated overnight at 4 °C. The plate was washed with washing buffer (0.1% Tween 20 in PBS) and then saturated for 1 h at RT with blocking buffer (5% dried skim milk in washing buffer). Next, dilutions (1:100 in blocking buffer) of the serum samples were added to each well and incubated for 2 h at RT. After washing, 100 µl/well of peroxidase-conjugated anti-mouse IgG (1/500) (Sigma) in blocking buffer was added and incubated at 37 °C for 1 h. For determination of the IgG1, IgG2a and IgG3 subclasses, ELISA plates coated as before were first incubated with an unconjugated rabbit anti-mouse IgG1, IgG2a or IgG3 (Bio-Rad) at 37 °C for 1 h and then with a peroxidase-conjugated goat anti-rabbit IgG (Sigma) overnight at 4 °C. After exhaustive washing, immune complexes were revealed by incubation with tetramethylbenzidine for 30 min at RT. The reaction was stopped by the addition of 50 µL/well 1 M H₂SO₄, and the absorbance was read with an ELISA reader (Multiskan ascent, Labsystem) at 450 nm.

The concentration of collagen in the supernatants was analyzed by ELISA. Treated and control cells were cultured with medium for 48 hours. Then the culture supernatants were collected, filtered and added to multi-well plates (Costar). After incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), followed by a peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations were performed for 1 h at 37°C and between each step washed three times with PBS wash buffer (PBS containing 0.05% Tween₂₀). Ortho-phenylene-diamine was used as a chromogen. Optical densities (OD) were measured at 492 nm in an Easy Reader (Bio-Rad).