Vector vip peroxidase substrate kit sk 4600

A focus-reduction microneutralization titer (FRμNT) assay was used to measure the neutralizing antibodies in serum specimens. First, Vero cells were seeded in 96-well plates and incubated for 20 h in a 37 °C incubator with 5% CO₂. Inactivated serum specimens were serially diluted and incubated with 100 focus forming units (ffu) of JEV at 37 °C for 1 h. The monolayer of Vero cells in the 96-well plate was infected with the serum-virus mixture at 37 °C for 1 h. The infected cells were overlaid with 1% methyl cellulose and 2% FBS in DMEM and incubated in a 37 °C incubator with 5% CO₂ for 30 h. The infected cells were then washed with PBS to remove 1% methyl cellulose, fixed with 75% acetone in PBS at room temperature for 20 min, and dried under a hood. The fixed cells were incubated with mouse anti-JEV polyclonal antibody at 37 °C for 40 min, and JEV-reactive mouse antibodies were detected by incubating with peroxidase-conjugated goat anti-mouse IgG (H + L) antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at 37 °C for another 40 min. The virus-infected foci were counted after staining with the Vector-VIP peroxidase substrate kit SK-4600 (Vector Laboratories, Burlingame, CA, USA). The cut-off value of FRμNT₅₀ was at the 1:10 serum dilution, and the foci count reduced by at least 50% of that in the virus-only control well.

The paraffin sections were dewaxed in xylene and rehydrated with distilled water. Following incubation with antibodies against either human CD3 (LN10) (Leica Biosystems) or human CD8 (SP16) (Biocare), the adjacent sections were stained with DAB Peroxidase Substrate Kit (SK-4100) and VECTOR VIP Peroxidase Substrate Kit (SK-4600) (Vector Laboratories). The positive cells were quantified using ImagePro Plus software (Media Cybernetics). All section stained TMA slides were examined by the reviewers who have no knowledge of any clinical data. Two cores of CT and IM regions were taken from the primary tumour as previously described.