3 3 diaminobenzidine tablets

To analyze gp83 epitope and His₆ expression, 5×10⁹ VPs/vector were boiled and resolved on SDS-PAGE gels, followed by transfer onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with 5% dry non-fat milk in Phosphate Buffered Saline with Tween 20 (PBST) buffer (1× PBS and, 0.05% Tween 20) for 1 hour. The membrane was incubated overnight at 4°C with His₆ MAb (1∶5,000 dilution in blocking buffer, GenScript, Piscataway, NJ), then washed, and incubated with HRP-conjugated goat anti-mouse antibody (1∶5,000; Millipore, Temecula, CA). The proteins were detected by using 3′3′-diaminobenzidine tablets (Sigma-Aldrich, MO) [30] (link).

Spleens were processed and stained as described previously (32 (link)). In short, spleens were frozen in liquid nitrogen and 5.5-μm-thick sections were cut and allowed to dry before fixation in acetone for 20 min at 4°C. In all cases, antibodies were added for 45 min of incubation at room temperature. Sheep anti-mouse IgD (Abcam) was detected with peroxidase-labeled donkey anti-sheep antibody (Jackson ImmunoResearch, Inc.) and developed with 3,3′-diaminobenzidine tablets (Sigma). CD3 and peanut agglutinin (PNA) were detected with rat anti-mouse CD3 (AbD Serotec), biotinylated rabbit anti-rat (Dako), and biotinylated rabbit anti-PNA (Vector Laboratories) antibodies. Biotinylated Ab binding signal was developed by using a streptavidin-biotinylated AP complex (Vectastain; Vector Laboratories) with Naphthol AS-MX phosphate, levamisole, and Fast Blue Salt.

For histological analysis, mice were killed 3, 7 and 14 days after treatment. The harvested wound tissues were fixed in 4% paraformaldehyde for paraffin embedding. Sections (6 μm thick) were used for various staining techniques: hematoxylin-eosin (HE) staining, Masson’s trichrome staining, Picrosirius red staining26 (link), and immunohistochemistry (IHC) staining for CD31 (ab28364, Abcam) and F4/80 (CI:A3-1, Abcam). For IHC, sections were deparaffinized, incubated for 20 min with 3% hydrogen peroxide to block endogenous peroxidase activity, incubated with Blocking One Histo (Nacalai Tesque) at room temperature, and then incubated with primary antibodies at 4 °C overnight. All sections were visualized using Histofine Simplestaining mouseMAX-PO (rabbit or rat) (Nichirei Biosciences, Inc.) and 3,3′-Diaminobenzidine tablets (Sigma-Aldrich). Stained sections were viewed by microscopy (Olympus IX83, Olympus), and digital images were acquired with a DP80 camera using the cellSens software (Olympus). Picrosirius red stained sections were observed using a polarized microscope. HE-stained sections were used to evaluate the re-epithelialization and granulation of wounds. Images were optimized globally and assembled into figures using Adobe Photoshop.

After 6 days of culture, bone slices were incubated in 0.5 N NaOH for 30 s, and cells were scraped off using a cotton swab. Bone slices were then incubated with 20 μg/mL peroxidase-conjugated wheat germ agglutinin (Sigma) in PBS for 30 min, washed with PBS three times, and exposed to 3,3′-Diaminobenzidine tablets (Sigma; D4168) for 15 min before washing. ImageJ (https://imagej.nih.gov/ij/) was used to quantify the pit area.

Hydrogen peroxide (3%) in 60% methanol was used to eliminate endogenous peroxidase activity. These sections were blocked with serum (Jackson ImmunoResearch) in 0.2% Triton-X in 1X PBS for 1 h at 37°C. Sections were then incubated overnight at 4°C with anti-progranulin antibody (R & D Systems; AF2420; 1:400) or anti-IBA1 (Wako; 019-19741; 1:500). Appropriate biotinylated secondary antibodies (Vector Labs) were then applied at 1:500 and incubated for 1 h at 37°C. Staining was performed using the avidin-biotin (Vector Labs) complex method, and tissue developed for 15 min using 3,3′-diaminobenzidine tablets (Sigma-Aldrich).

Protein expression was analyzed from lysates of HEK293 cells infected with Ad5-CMV-ASP-2 and Ad48-CMV-ASP-2. The other modified vectors, Ad5-pIX-ASP-C, Ad5-pIX-gp83, Ad48-pIX-ASP-C, and Ad48-pIX-gp83 (5 × 10⁹ VPs/vector) were analyzed for the protein of interest incorporated within the capsid. The lysates and vectors were denatured by boiling and resolved on SDS-PAGE gels, followed by transfer onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with 5% dry non-fat milk in PBST for 1 h. Thereafter, the membranes were incubated overnight at 4 °C with His₆ MAb (1:5000 dilution in blocking buffer; GenScript), or FLAG-HRP antibody. After overnight incubation, the membranes were washed, and the His₆ membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:5000; Millipore, Temecula, MA, USA). The proteins were detected by using 3′3′-diaminobenzidine tablets (Sigma-Aldrich, St. Louis, MO, USA) [56 (link)].

To analyze both the His₆ and V3 presentations on viral capsid, recombinant vectors were boiled and resolved on SDS-PAGE gels, followed by transfer and blocking on PVDF membranes. Blotting was performed with anti-penta-His MAb (1:2,000; Qiagen, CA), anti-gp120 (902) MAb (1:1,000; NIH AIDS Research & Reference Reagent Program: 522)
[30 (link)] and anti-IIIB-V3-21 MAb (1:500; NIH AIDS Research & Reference Reagent Program: 1725), respectively, followed by secondary incubation with HRP-conjugated goat anti-mouse antibody (1:5,000; Millipore, MA). The proteins were detected by using 3’3’-diaminobenzidine tablets (Sigma-Aldrich, MO)
[34 (link)].