Goat anti mouse secondary antibody

Hippocampi were thawed and homogenized in lysis buffer (200 mM HEPES, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM Na2VO4, 150 mM NaCl, and 50 mM NaF) supplemented with protease inhibitor (Roche). Cells were incubated for 1 h at 4°C. Proteins were cleared by centrifugation at 14,000 × g for 20 min at 4°C. Protein concentrations were quantified utilizing the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific). 25 μg protein from each sample was resolved in SDS-PAGE. Nitrocellulose transferred membranes were blocked for 1 h with PBS 0.1% Tween 20 with 5% bovine serum albumin and were probed with goat anti APOE antibody (1:10,000, Chemicon) overnight in 4°C and with goat anti ACTIN antibody (1:5000, MAB1501, Milipore) for 1 h at room temperature, followed by incubation with Goat anti Mouse secondary antibody (1:5,000, Licor) for 1 h at room temperature. Visualization and analysis of band intensities were performed using the Odyssey system (Licor) and the Image Studio Lite 5.2 software. For each sample, APOE results were normalized to ACTIN.

Polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4°C with antibodies specific for NF-κB p50 and NF-κB p65 (Santa Cruz Biotechnology). Samples were then washed in Tris-buffered saline with Tween-20 and labeled further with a goat anti-mouse secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA). Labeled protein bands were detected using an Odyssey infrared imaging system (LI-COR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference control.

C. jejuni strains expressing GFP were grown as for fluorescence measurement described above and whole cell lysates prepared after normalizing by OD₆₀₀. Western blots were performed using mouse anti-GFP antibody (Sigma) and goat anti-mouse secondary antibody (Li-Cor).

After 24 h of reperfusion, rat brain tissue from the ischemic penumbra was collected and lysed in high-efficiency RIPA lysis buffer containing PMSF. The protein concentration was determined using a BCA assay. Equal amounts of protein were loaded and separated on 10–12% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes in Tris-glycine transfer buffer. The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with a primary antibody against β-actin (1:1000), β-tubulin (1:1000), Iba1 (1:1000), CD40 (1:1000), CD68 (1:1000), CD206 (1:1000), GPER (1:1000), P65-NF-κB (1:1000), p-P65-NF-κB (1:1000), ERK (1:1000), or p-ERK (1:1000) at 4 °C overnight. The membranes were then incubated with a goat anti-mouse secondary antibody (1:5000; LI-COR, USA) or goat anti-rabbit secondary antibody (1:5000; LI-COR, USA) at room temperature for 1 h. The bands corresponding to the antigen–antibody complexes were detected using Super Signal West Pico Chemiluminescent Substrate and visualized with an Amersham Imager 600 system.

Neutrophils were infected with B. anthracis at an approximate MOI of 1 as described above. After 1 h of infection, plates were spun down at 290 × g for 5 min and supernatants dotted on nitrocellulose membranes (BioRad) and allowed to air dry. Membranes were blocked in PBS blocking buffer (Li-Cor) for 1 h, then incubated overnight with 1:1,000 dilution anti–LL37 (Santa Cruz Biotech) or anti α-defensin (Santa Cruz Biotech). Membranes were then washed with 0.1% PBS-Tween and incubated for 1 h with goat anti-mouse secondary antibody (Li-Cor). After washing with 0.1% PBS-Tween, membranes were imaged on Li-Cor Odyssey CLx. Densitometry analysis was performed using ImageJ software (National Institutes of Health). For elastase activity assay, supernatants from infected neutrophils were collected as described above. Supernatants were incubated with 1 mM of the colorimetric elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (p-nitroanilide) (Sigma). Elastase activity was measured after 30 min on a PerkinElmer EnSpire Alpha plate reader at an optical density of 405 nm (OD₄₀₅).

Protein extraction and quantification were performed as described (11 (link)). Protein (75 μg) was loaded into each lane of a Mini-PROTEIN 4–15% gel (Bio-Rad). The semidry transfer was performed onto a PVDF membrane using TRANS-BLOT SD (Bio-Rad). Membrane was blocked in blocking buffer (5% milk in Tris-buffered saline, 0.1% Tween 20 solution (TBST) for 60 min at room temperature. Primary antibody was used against V5 epitope (Monoclonal V5 epitope tag antibody (Invitrogen, catalogue no. R960-25) at a dilution of 1:1000 in TBST containing 5% BSA and incubated at 4 °C for overnight. The primary antibody was then removed, and the membrane was washed three times with TBST for 10 min each. The goat anti-mouse secondary antibody (LI-COR Biosciences, catalogue no. 926-322-10) was used at a dilution of 1:10,000 in TBST containing 5% milk. Membranes were incubated in secondary antibody for 60 min at room temperature. Secondary antibody was removed, and the membrane was washed three times with TBST for 10 min each. Finally, the membrane was visualized using the Odyssey FC imaging system. Glucose-6-phosphate dehydrogenase (G6PD) was used as the loading control. The same procedure as above was employed for G6PD; primary antibody was anti-G6PD (Sigma-Aldrich, catalogue no. HPA000-834), and secondary antibody was goat anti-rabbit (LI-COR Biosciences, catalogue no. 926-68071).