Ab6858

Immunohistochemistry was performed to detect the expression of SENP1, Ki67, and p-STAT5. The detailed process has been described in our previous article [17 ]. In brief, the formalin-fixed, paraffin-embedded specimens were sliced into 4 µm tissue sections, then depolished with xylene and rehydrated with an alcohol solution. After antigen retrieval, the tissue sections were incubated with anti-SENP1, anti-Ki67, or anti-p-STAT5 primary antibody for 24 hours (h) at 4 °C, and then washed with PBS and incubated with peroxidase-conjugated goat anti-human/mouse secondary antibody (1:200 dilution, #ab6858 and #ab150113, Abcam, USA) for 1 h at room temperature. Five fields were randomly selected from each section under a light microscope (100×, BX51, Olympus, Japan) fitted with a digital camera (DP72, Olympus, Japan). Image-Pro Plus 6.0 software was used to quantitively analyze the images. Quantification of SENP1, Ki67, and p-STAT5 was determined by mean of integrated optical density (IOD).

MaxiSorp 96-well flat-bottomed plates were coated with 50 μl of 125 nM recombinant human or mouse ACE2 protein in PBS at room temperature for 1 h. All washes were performed three times using PBS and 0.1% Tween-20 and all incubations were performed for 1 h at room temperature. Coated plates were washed and blocked by incubation with 4% skim milk solution. Plates were washed and then incubated with 50 μl of 125 nM of anti-ACE2 monoclonal antibodies. The plates were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibodies (ab6858, Abcam, 1:5,000). After a final wash, 50 μl of azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS liquid substrate; Sigma-Aldrich) was added and incubated in the dark at room temperature for 20 min and 50 μl of 1% SDS was used to stop the reaction. Absorbance was read at 405 nm and all samples were done in duplicate.

Nunc MaxiSorp plates (Thermo Fisher Scientific, Waltham, MA) were coated with ZIKV E or E domain III protein (1 µg/mL) and incubated at 4°C overnight. After blocking for 2 h, the plates were washed six times with phosphate-buffered saline (PBS) and transfected with antibody supernatants at a 1:2 dilution with blocking buffer or a serial dilution of purified mAbs. Secondary antibody (goat anti-human IgG; ab6858; Abcam, Cambridge, UK) was applied at a 1:5000 dilution in blocking solution and then the plates were incubated at room temperature for 1 h, and TMB substrate. Absorbance values were determined at 450 nm using a BioTek plate reader (BioTek Instruments, Winooski, VT).

Microtiter plates were coated with purified BpOmpW at 0.5 μg/ml in sodium bicarbonate buffer (pH 9.4) at 4°C overnight, after which the coating solution was removed and the plates blocked with 1% FBS (7524; Sigma) solution in PBS at room temperature for 1 h. The wells were washed three times with 0.05% Tween 20 in PBS using a plate washer (5165040; Thermo Scientific). All serum samples were serially diluted (fivefold) in PBS containing 1% FBS and 100 μl added to the wells in triplicate at room temperature (RT) for 2 h. The plates were then washed three times with 0.05% Tween 20 in PBS, as described above, before the addition of anti-mouse immunoglobulin G (IgG), IgG1, or IgG2a–horseradish peroxidase (HRP)-conjugated antibodies (ab97023, ab97240, ab97245, respectively; Abcam, Cambridge, UK) at RT for 1 h. Then, the TMB (3,3′,5,5′-tetramethylbenzidine) substrate was added and incubated until the color developed, at which time the reactions were stopped with 2 M sulfuric acid and the plates read at 450 nm. For the detection of BpOmpW-specific IgG in human plasma, the same protocol was applied using anti-human IgG–HRP antibody (ab6858; Abcam) in place of anti-mouse IgG antibodies. All the HRP-conjugated antibodies were diluted 1:5000 in PBS containing 1% FBS. The color intensity at OD₄₅₀ correlated with the amount of specific antibodies in the sera.