Ab150076

The pcDNA3.1(−)-TIM-1 and pVAX1-NS1′ were preserved in our laboratory. JEV E gene was inserted into p3×FLAG-CMV-7.1. hTIM-D114A-His, hTIM-N115A-His, and hTIM-MD were cloned into pcDNA3.1 (+). hTIM-IgVD was cloned into pCAGGS. hTIM-ECD was inserted into prokaryotic expression vectors pET-28a (+) and pGEX. Mouse anti E monoclonal antibody was kept by our lab. Mouse anti-human TIM-1 monoclonal antibody (MAB1750, R&D systems, Minneapolis, MN, USA) and goat anti-TIM-1 polyclonal antibody (AF1750, R&D systems, Minneapolis, MN, USA) were used for detecting the TIM-1 protein in Western blot and IFA. Mouse anti-His antibody (66005-1-Ig, Proteintech, Rosemont, IL, USA) was used for the enrichment of TIM-1-His protein. Mouse anti-GAPDH polyclonal antibody (sc-25778, Santa Cruz, Dallas, TX, USA) was used for Western blot. Rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex, Irvine, CA, USA), rabbit anti-JEV NS3 polyclonal antibody (GTX125868, GeneTex, Irvine, CA, USA), and rabbit anti-JEV polyclonal prM antibody (GTX131833, GeneTex, Irvine, CA, USA) were used to detected the JEV E, NS3 and prM protein, respectively, in Western blot analyses. Donkey anti-goat IgG-Alexa Fluor488 (ab150129, Abcam, Cambridge, UK), donkey anti-rabbit IgG Alexa Flour 594 (ab150076, Abcam, Cambridge, UK), and donkey anti-mouse Alexa Flour647 (ab150107, Abcam, Cambridge, UK) were used for IFA.

Immunofluorescence staining was performed to detect phosphorylated H2A histone family member X (γ-H2AX) in the paraffin sections and RLE-6TN cells. The samples were treated with rabbit anti-γ-H2AX (1:300, ab81299, Abcam) primary antibodies overnight at 4 °C, washed and followed by incubation with the secondary antibody Donkey Anti-Rabbit IgG H&L Alexa Fluor^® 594 Conjugate (ab150076, Abcam) for 4 hours at room temperature. Double immunofluorescence staining was used to detect the expression of γ-H2AX in AECIIs and the combination of primary antibodies used was rabbit anti-γ-H2AX (1:300, ab81299, Abcam) and mouse anti-p180 (ribosome-binding protein 1) (1:100, ab24751, Abcam). The combination of secondary antibodies used was Donkey Anti-Rabbit IgG H&L Alexa Fluor^® 594 Conjugate (ab150076, Abcam) and Donkey Anti-Mouse IgG H&L Alexa Fluor^® 488 Conjugate (ab150105, Abcam).

For immunofluorescence assays, dissected mosquito ovaries were dissected into phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at RT. To block non-specific binding, ovaries were incubated for 2 h at RT with 3% bovine serum albumen in PBS with 0.1% Tween 20. Following blocking, Cas9 protein was detected using rabbit anti-Cas9 polyclonal antibody (Abcam ab204448) diluted 1:500 in PBS with 0.1% Tween 20 and incubated for 1 h at RT or overnight at 4 °C. After incubation, samples were washed three times with 0.1% Tween 20 in PBS, and the primary antibody labeled with 1:500 anti-rabbit Alexa Fluor 594 secondary antibody (Abcam ab150076). After three additional washes, ovaries were air-dried and slide mounted with ProLong Gold Antifade Reagent (Molecular Probes) and visualized using epifluorescent microscopy.

The organoids were then formalin fixed and paraffin embedded. Sections were deparaffinized with xylene, treated with antigen retrieval solution (1 g NaOH, 2.1 g citric acid in 1 L of H₂O) for 20 min in steam, cooled to room temperature, permeabilized with PBS-0.2% Triton for 15 min, and blocked with 10% donkey serum in PBS. Sections were stained with monoclonal rabbit antithyroid transcription factor 1 (TTF1, ab76013; Abcam, Cambridge, MA) or rabbit anti-CC10 (ab40873, Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 594-conjugated donkey antirabbit secondary antibody (ab150076, Abcam, Cambridge, MA, USA) at 1:500, goat anti-GFP (ab5450; Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 488-conjugated donkey anti-goat secondary antibody (ab150129, Abcam, Cambridge, MA, USA) at 1:500. Images were taken at 10×, 20×, and 40× with an Olympus FV1000 confocal microscope. Image analysis were performed using ImageJ software.

Colon samples were sectioned at a thickness of 4 μm and pretreated with citrate buffer solution (pH 6.0) for 30 min at 95°C. After blocking in 5% BSA for 30 min, sections were incubated with primary antibodies (goat anti-RARα, Abcam ab28767, UK; rabbit anti-CBP, Novus NB100-91721, USA; mouse anti-β-Tubulin III, Santa Cruz sc-80005, USA; rabbit anti-NeuN, Millipore MABN140, USA; mouse anti-NeuN, Millipore MAB377) overnight at 4°C. Sections were washed with PBS and incubated in corresponding secondary antibodies (donkey anti-goat Alexa Fluor®594, Abcam ab150129; donkey anti-rabbit Alexa Fluor®594, Abcam ab150076; donkey anti-rabbit Alexa Fluor®488, Abcam ab150073; chicken anti-mouse Alexa Fluor®488, Invitrogen 1696214, USA; goat anti-rabbit Dylight ®488 Abbkine A23220,USA; goat anti-mouse Dylight®594, Abbkine A23410) for 1.5 h at 22°C.