Detection again followed the manufacture's method, in brief, the membrane was blocked, washed, and incubated with streptavidin conjugated HRP.
Surgical method in brief: The same team of surgeons performed each operation (JSa, SRä, INo).
Calcium methoxyethoxide (CME) was prepared following the method in literature.[ 7b, 21] In brief, calcium granules (1 g) were reacted with 2-methoxyethanol (24 mL) under an argon atmosphere at 80 °C for 24 h.
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An improved bi-conjugate residual (BiCR) method (IBiCR method, in brief) for solving large sparse linear systems with unsymmetrical coefficient matrices was proposed for distributed parallel environments.
Three independent assay runs were performed for each sera pool per virus by a single technician using the method detailed below: In brief, serial 10-fold dilutions of the serum pools were mixed with a constant challenge dose of each respective JEV (45 60 plaques per well) and inoculated in duplicate, onto wells of a 6-well plate of confluent LLC-MK2 cells.
The TCID50 (50% infectious dose in tissue culture) of the virus was estimated using a previously described method., In brief, 10-fold serial dilutions of allantoic fluid containing the virus were inoculated into Madin-Darby canine kidney cells (MDCK ATCCC No. CCL-34) in a 96-well culture plate and incubated for 3 d at 37°C in a 5% CO2 humidified atmosphere.
The methods are described in brief here and in more detail in the Technical Appendix.
As described in the 'Methods' section, a brief rinse in low-surface-tension fluid was used to reduce the probability of pattern collapse.
For B19V nPCR and qPCR the methods are described in brief as follows: DNA extracted from EMBs, PBMCs and serum, respectively, was amplified using B19V-specific primers yielding a product of 290 bp in the first and of 173 bp in the second round.
