Vectastin abc kit

Lung sections (sham 0 and 12 and CLP 0 and 12, n = 3 in each group) were deparaffinized and hydrated. To examine their histological features, the lungs were stained with hematoxylin and eosin (H&E). After blocking of endogenous peroxidase, antigen retrieval was performed in a high-temperature Tris-citrate buffer (pH 7.2). The rabbit polyclonal anti-SOD3 (Santa Cruz Biotechnology) (diluted 1:1,600) was used as the primary antibody. The Vectastin ABC Kit (Vector Laboratories, Burlingame, CA, USA) was used as the secondary antibody, and 3,3-diaminobenzidine (DAB, Sigma, St. Louis, MO, USA) was used as the chromogen. Thereafter, the sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). In the negative controls, the first antibody was omitted from the procedure, and the tissues were incubated with bovine serum albumin (BSA) instead.

The presence of neutralizing antibodies in the sera of vaccinated mice were determined by FRNT as previously described^{56 (link)}. Briefly, 2-fold serial dilutions of sera (starting dilution of 1:800) were incubated with 25 FFU of rZIKV for 3 h at 37 °C, and used to infect subconfluent (80%) monolayers of Vero cells in 96-well plates. After 90 min of viral absorption at 37 °C, virus-sera mixture was removed, and the cells overlaid with 100 μl of growth medium containing 2% FBS and 1% Avicel. Cells were fixed at 36 h post-infection with 4% paraformaldehyde and prepared for immunostaining assay using 1 μg/ml of mAb 4G2. Viral plaques were visualized using Vectastin ABC kit and DAB HRP substrate (Vector Laboratories Inc.), following the manufacturer’s instructions.

Hearts were perfused ex vivo via the aorta with 0.9% NaCl before fixation in 3.7% formaldehyde solution overnight. After embedding in paraffin, 5 μm sections were cut and stained for 60 min at room temperature in Sirius Red solution, i.e. 0.1% Direct Red 80 (Sigma, Taufkirchen, Germany) in saturated aqueous picric acid (Morphisto, Frankfurt, Germany) adjusted to pH 2.0 with sodium hydroxide [24 (link)]. The slides were tipped four times in 0.5% acetic acid solution and incubated 5 min each in ethanol and in isopropyl alcohol. After two further incubations for 10 min in xylole, the slices were embedded in Histokitt (Carl Roth) and recorded at 20x magnification using a slide scanner (Olympus, Hamburg, Germany). The extent of fibrosis based on the amount of collagen was quantified in two complete heart sections using cellSens Dimensions software (Olympus) by a scientist blinded to the genotype of the mice.
Kidneys were immersion-fixed in 3.7% formaldehyde solution overnight and embedded in paraffin. Immunohistochemical demonstration of eGFP was performed on 5 μm sections using a rabbit anti-eGFP polyclonal antibody (Abcam, Cambridge, UK) and the Vectastin ABC kit (Vector Laboratories, Burlingame, CA) using DAB (3,3’-diaminobenzidine tetrachloride) as a substrate. Quenching of endogenous peroxidase activity was achieved by treating the sections with 3% H₂O₂ in ethanol.