Peroxidase conjugated goat anti mouse igg

Cell lysates were electrophoresed through polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated with polyclonal antibodies specific for NF-κB p65 (Cat. ab72555; abcam; UK), TGFß1 (cat. sc-146; Santa Cruz Biotechnology, Inc; Germany), IRF6 (cat. sc-98829; Santa Cruz Biotechnology, Inc; Germany), TNF-α (cat. LS-C43037; LSBio; Germany) and ß-actin (cat. sc-47778; Santa Cruz Biotechnology, Inc; Germany), and then revealed with secondary antibody. As a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit (cat. Sc-2004; Santa Cruz Biotechnology, Inc; Germany) was used for the primary antibody of NF-κB p65, IRF-6, and TNF-α; whereas, peroxidase-conjugated goat anti-mouse IgG (cat. Sc-2005; Santa Cruz Biotechnology, Inc; Germany) was used for ß-actin primary antibody. Mouse polyclonal anti-ß-actin antibody was used to correct minor differences in protein loading. Finally, the specific signals were detected by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (cat. 34077, Thermo Scientific, Germany). Images were acquired by Quantity One 1-D analysis software (Bio-Rad, Germany).

Spleens from naive or mice immunized intraperitoneally with M2e peptide (50 μg) in the presence of 200 μg of pJAK2(1001–1013), or control peptide (scram) were harvested after 4 weeks and homogenized to single-cell suspension. Splenocytes (10⁵ cells/well) were incubated with medium alone or medium containing M2e peptide (50 μg) at 37°C for 72 h. CellTiter Aqueous One Cell Proliferation Assay reagent (Promega) was added and the absorbance was measured.
Blood was drawn from naïve mice or those immunized with M2e peptide in the presence of pJAK2 or control peptide after 2 or 3 weeks. Microtiter plates coated with M2e peptide (10 μg/well) were blocked with PBS containing 5% FBS for 2 h at room temperature. Mouse serum was serially diluted (0.1 ml) in PBS containing 0.1% Tween 20 (wash buffer). Plates were incubated for 2 h at room temperature and washed three times with wash buffer. Peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology), diluted in a volume of 0.1 ml, was added to each well, incubated for 1 h, and washed five times with wash buffer. o-Phenylenediamine (OPD) (0.1 ml) was added and incubated for 15 min. The reaction was stopped by addition of 50 μl 3 N HCl. The OD at 490 nm was determined using a microtiter plate reader.

For the detection of TIMP and MMP protein expression 15 μg of total protein was separated on a 10 % polyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Schwalbach, Germany). After blocking in PBS supplemented with 5 % skim milk (Sigma-Aldrich) and 0.05 % Tween 20 (Sigma-Aldrich) membranes were incubated overnight at 4 °C with one of the following primary antibodies at the indicated dilutions: MMP2 (1:500) (Abcam, Cambridge, United Kingdom), MMP9 (1:500) (Abcam) TIMP1 (1:250) (Abcam), TIMP2 (1:250) (Abcam), TIMP3 (1:250) (Abcam). After washing three times in PBS containing 0.1 % Tween 20 membranes were incubated for 1 h at room temperature with 5000-fold diluted peroxidase conjugated goat anti-mouse IgG (Santa Cruz). Proteins recognized by the antibody were visualized with luminata forte western blotting substrate (Millipore) according to the manufacturer’s instructions. Signal intensities were quantified by densitometry using Bio-1D software version 15.01 (Vilber Lourmat, Eberhardzell, Germany).

For western blot analysis, whole retinas were used immediately or frozen at −70 °C until use. Retinal tissues were homogenized in a glass-Teflon Potter homogenizer in PRO-PREP^TM lysis buffer (iNtRoN Biotechnology, Inc., Seoul, Korea). Next, 10 µg of each sample was separated on a 10% polyacrylamide mini gel. After protein transfer, the membranes were blocked for 1 h at room temperature in Tris-buffered saline–Tween-20 solution [TBS-T; 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20] containing 5% non-fat dry milk. After blocking, the membranes were incubated overnight at 4 °C with a primary antibody against GFAP (1:3000; Cell Signaling Technology, Inc., Danvers, MA, USA), Iba1 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and β-actin (1:10,000; Sigma, MO, USA). After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with a peroxidase-conjugated goat anti-mouse IgG (1:3000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or peroxidase-conjugated goat anti-rabbit IgG (1:3000; Cell Signaling Technology, Inc., Danvers, MA, USA) in TBS-T containing 5% non-fat dry milk. The signals were visualized by enhanced chemiluminescence and quantified using an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).

Cells were ruptured on ice with 1x RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA), containing protease inhibitor (Roche) and phosphatase inhibitor (Thermo Fisher Scientific Inc.). Cell lysates were clarified by centrifugation, and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in gels were transferred onto polyvinylidine (PVDF) membranes (Bio-Rad), and these were blocked with 5% BSA (Gibco) in PBS-T. Membranes were then incubated with antibodies against TRIF (Abcam), Myd88 (Cell Signaling Technology), TLR4 (Invitrogen), phospho-TRAM (MyBioSource, San Diego, CA, USA), TRAM (R&D Systems), phospho-TIRAP (Y86; Abcam), TIRAP (Abcam), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), phospho-p65 (Ser536; Cell Signaling Technology), and p65 (Enzo Life Sciences, Inc.) overnight at 4°C. After washing with PBS-T, membranes were stained with peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) or peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). Target proteins were then detected using the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore).