Hrp labeled goat anti rabbit igg

After the MWM test, three rats from each group were sacrificed for Western blot analysis to collect the hippocampal tissue. The tissue was homogenized in RIPA lysis buffer (1:5, w/v). Protein quantification was performed using a BCA protein assay kit. Accurately weighed 27 μg of protein was used for electrophoresis. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% fat-free milk powder in TBST buffer for a duration of one hour at room temperature, followed by incubation with primary antibody: p-p38 mitogen-activated protein kinase (1:1000), extracellular signal-regulated kinases (ERK) (1:1,000), brain-derived neurotrophic factor (BDNF) (1:2,000), p-cAMP-response element binding protein (p-CREB) (1:1,000), and p-extracellular regulated protein kinase (p-ERK) (1:2,000), acquired from Abcam, USA, for 12 h at 4°C. Subsequently, incubation of HRP-labeled goat anti-rabbit IgG (Jackson 111-035-003, 1:10,000) with the membrane was conducted for 60 minutes at room temperature. Finally, the visualization and scanning of the blots were carried out utilizing enhanced chemiluminescence (ECL) Western blot detection (Millipore WBKLS0500).

Quantifications of Trx and TrxR proteins were conducted by Western blot analyses as previously described, with minor modifications [32 (link), 43 (link)]. Briefly, equal amounts of protein (5 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane at 250 mA for 90 min. Membranes were blocked at 4°C overnight with 5% dry skim milk in 0.05 M Tris-buffered saline pH 7.6, containing 0.05% Tween-20 (TBS-T). Subsequently, the membranes were incubated with Trx or TrxR primary antibodies, which were generously provided by Dr. S.G. Rhee (Ewha Women University, Seoul, Korea). After washing with TBS-T, the membranes were incubated for 1h at room temperature with HRP-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA). Next, the membranes were washed with TBS-T and developed using light sensitive film (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and the chemiluminescence detection kit for HRP (EZ-ECL; Biological Industries, Beit-Haemek, Israel) according to manufacturer's instructions. Band intensities were quantified using IMAGE J 1.32 software (NIH, USA).

ELISA experiments with His-tagged SOSIP trimers were performed essentially as described before^{13 (link),51 (link)}. Purified proteins (1 µg/mL) were diluted in TBS and immobilized on 96-well Ni-NTA functionalized ELISA plates (Qiagen) by a 2 h incubation at room temperature. Following a double wash step with TBS to remove unbound trimers, serial dilutions of test antibodies in TBS/2% skimmed milk were added and incubated for 2 h. After 3 washes with TBS, HRP-labeled goat anti-human IgG (Jackson Immunoresearch) diluted 1:3000 in TBS/2% skimmed milk was added and incubated for 1 h, followed by 5 washes with TBS/0.05% Tween20. A developing solution (1% 3,3’,5,5’-tetramethylbenzidine (Sigma-Aldrich), 0.01% H₂O₂, 100 mM sodium acetate and 100 mM citric acid) allowed the colorimetric reaction, which was stopped by the addition of 0.8 M H₂SO₄. Finally, color development (absorption at 450 nm, OD₄₅₀) was measured to obtain the different binding curves.
Endpoint antibody titers from rabbit sera samples were determined using Ni-NTA His-Tag-capture ELISA similarly as above described^{6 (link),51 (link)}. However, in this case, sera was diluted in TBS/2% skimmed milk/20% sheep serum and the secondary antibody was an HRP-labeled goat antirabbit IgG (Jackson Immunoresearch).