Anti mouse igg peroxidase conjugate

L1:P18I10 VLPs purified from both methods were treated or un-treated with 20 mM DTT for 15 min and were then filtered out through 1000 kDa (SARTORIUS, Göttingen, Germany) or 100 kDa (Amicon) molecular weight cutoff (MWCO) ultrafiltration devices. The retentates were reconstituted to the original volume and collected from the filter device sample reservoir, while the filtrates were collected at the bottom of the centrifuge tube. The L1 signal was measured by using dot blot probed with anti-HPV16 L1 mAb and detected by anti-mouse IgG-peroxidase conjugate (Sigma-Aldrich). Images were acquired using Odyssey Fc Imaging System at a chemiluminescence channel.

Cells from both monolayer cultures and NS, after 0–45 days of culture, were collected in a lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Triton X-100, 0.5 mM EDTA, 0.1% SDS, 50 mM Tris-HCl, pH 7.6), containing protease inhibitor cocktail (Sigma-Aldrich), and 17.4 µg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein extracts were quantified by Bradford Protein Assay. For Western-blot analysis, 50 µg of total proteins were resolved on denaturating polyacrylamide gels (SDS-PAGE): 10% (Msi-1, βIII-Tubulin) and 8% (Nestin) and transferred to polyvinylidene difluoride (PVDF; Immobilon P, Millipore, USA) membrane by electroblotting. Membranes were blocked with PBS-T (PBS buffer saline with 0.1% Tween-20) containing 5% BSA and then incubated overnight with primary antibodies. After washing for 3×5 min., membranes were probed 1 h at room temperature with secondary antibody (anti-mouse IgG peroxidase conjugate, Sigma, 1∶10,000). The revelation was performed by chemiluminescence.
The signals were quantitated by densitometric scanning (ChemiDoc documentation system/QuantityOne quantitation software; Bio-Rad Laboratories, Hercules, CA, USA).

Immunoblotting and immunofluorescence staining were performed to determine the levels and distributions of target proteins. Frozen sections were cut after fixation with 4 % formaldehyde at room temperature for 10 min. Paraffin sections were prepared for hematoxylin and eosin (HE) staining to evaluate the morphological changes in transgenic mouse retinas. The antibodies and working dilutions used for immunofluorescence analysis and immunoblotting were as follows: rabbit anti-SIRT1 (1:500–2,000; #1104-1, Epitomics), anti-rhodopsin (1:500–2,000; #sc-57433, Santa Cruz), rabbit anti-GNAT1 (1:200–1,000; # sc-389, Santa Cruz), goat anti-CNGA1 (1:500–1,000; #sc-13694, Santa Cruz), goat anti-PDC (1:500–1,000; #sc-18413, Santa Cruz), anti-PDE6b (1:200–1,000; #sc-30717, Santa Cruz), anti-actin (1:2,000–10,000; #A5228, Sigma-Aldrich). Corresponding IgG antibodies conjugated with Alexa Fluor^® dye (594 or 488; Invitrogen) were used as secondary antibodies for the immunofluorescence analysis. In immunoblotting, anti-mouse IgG peroxidase conjugate (1:50,000; #a2304, Sigma-Aldrich), goat anti-rabbit IgG peroxidase conjugate (1:50,000; #a9169, Sigma-Aldrich), and rabbit anti-goat IgG peroxidase conjugate (1:80,000; #a5420, Sigma-Aldrich) were used as probes for the proteins of interest.

The Aβ species in the transgenic C. elegans strains was identified by immunoblotting using a Tris-Tricine gel and the standard Western blotting protocol except that the polyvinylidene difluoride membranes were boiled for 5 min after the transfer. After the experimental treatments, the worms were collected, washed with M9 buffer, flash frozen in liquid nitrogen, sonicated in the lysis buffer (62 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol (v/v), 10% glycerol (v/v), 2% SDS (w/v), and 1X protease inhibitor cocktail), and heated with sample buffer containing 5% β-mercaptoethanol. After heating with the sample buffer, the proteins were cooled and equal amounts of the total protein (60-80 μg) were loaded in each lane. Amyloid protein species were detected with 6E10 monoclonal antibody (1:750, Covance); secondary anti-mouse IgG peroxidase conjugate (1:5000; Sigma). Tubulin was detected with anti-tubulin antibody (1:2000, Abcam). The mean densities of β-amyloid reactive bands were analyzed by ImageJ (National Institutes of Health, USA).

Equal amounts (500 ng) of HPV16 L1 protein (Abcam), purified L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and then transferred to a PVDF membrane (Millipore, Burlington, MA, USA) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000, anti-HIV-1 gp120 V3 loop mAb (NIBSC, EVA3012) at a dilution of 1:40 and HIV1 gp41 (2F5) mAb (NIBSC, ARP3063) at a dilution of 1:4000, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The Western ECL substrate kit (BIO-RAD) was used for signal development. The blot images were acquired by using Odyssey Fc imaging system at a chemiluminescence channel.

Animals received three immunizations with 20 day intervals between doses, and the serum IgG was obtained before each immunization. In brief, 4 μg/mL of hybrid MSP1a/OMP7/8/9 were sensitized overnight at 4 °C in 96-well microplates (Nunc MaxiSorp^®). After blocking for 1 h with 2% casein-PBS buffer (blocking buffer) at room temperature, the plates were washed (4 times) with 0.05% Tween-20-PBS buffer. The serum was diluted at a ratio of 1:50 in the blocking buffer and incubated for 16 h at 4 °C. After washing, the anti-mouse IgG peroxidase conjugate (Sigma, USA) was used at a dilution of 1:10 000 in the blocking buffer. After washing, TMB (BD Biosciences, USA) was added for 15 min and blocked with 2.5 M H₂SO₄. For immunogenicity evaluation, the optical density (OD) was measured at 450 nm and the reactivity of sera from non-immunized mice were used to determine the cut-off for anti MSP1a/OMP7/8/9. First, the mean was calculated with OD values of sera collected of each animal of three groups before to start the immunization (N = 15). The variance among the OD values was estimated by standard deviation (SD). The cut-off was determined by equation cut-off = mean OD + 2 × SD. The values above the cutoff were considered positive.