Secondary goat anti rabbit igg

Vero cells were uninfected or infected at an MOI 1 with either VC2 or VC2-OVA for 24 and 48 h in a six well plate. Adherent cells were washed 3x in PBS followed by lysis in 200 μl of NP40 lysis buffer with protease/phosphatase inhibitors. Twenty microliters of whole cell lysate were then mixed with Laemmli sample buffer (Bio-Rad) and 1 μl of β-mercaptoethanol to a final 1x concentration. These mixtures were then boiled at 100°C for 10 min and cooled on ice before loading into a 12% Mini-PROTEAN TGX precast gel (Bio-Rad) and separated for 1 h at 100 V in 1x Tris-Glysine-SDS buffer (Bio-Rad). Separated protein was then transferred to a nitrocellulose membrane in 1x Tris-Glysine + 20% methanol (Bio-Rad). The membrane was then blocked for 30 min in 5% BSA in PBS-T. Rabbit anti-VP26 (Kind gift from Prashant Desai, Johns Hopkins), was diluted 1:1,000 in 5% BSA PBS-T and applied to the membrane and incubated overnight at 4°C while rocking. The next day, the membrane was then washed 3x with PBS-T and secondary goat anti-Rabbit IgG (Abcam: ab6721) diluted 1:1,000 in 5% BSA PBS-T applied to the membrane and incubated at room temperature for 1 h. The membrane was then washed 3x in PBS-T and visualized using ECL Western Blot Substrate (Pierce) and exposure film.

The slides containing sheared villi crypts and the enteroid samples were wetted with phosphate buffered saline (PBS) then treated with Cytovista 3D clearing agent (www.thermofisher.com) for 10 min followed by an additional washing step with PBS. The tissues were then permeabilized with 0.5% Triton X-100, and blocked with 10% goat serum (Sigma-Aldrich, Inc.) in PBS for an hour. The tissues were rinsed with PBS once to remove the blocking solution and the excess PBS was removed by vacuum aspiration. A rabbit anti-Tβ4 antibody (https://www.abcam.com) prepared in PBS containing 0.1% bovine serum albumin (BSA) placed over the tissues and incubated overnight at 4 °C in a humidified chamber. The following day, the slides were rinsed 3 times with PBS and probed with a secondary goat anti-rabbit IgG conjugated to Alexa fluor 488 (Abcam) for 1 h at room temperature in dark. After 3 further wash steps with PBS, the tissues were counterstained with 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI; 0.2 µg/ml PBS) and mounted with ProLong gold anti-fade reagent (www.thermofisher.com). F-actin was stained using Acti-stain (Alexa 550 labeled phalloidin, www.cytoskeleton.com) following the recommended protocol of the manufacturer. The images were photographed with an Olympus BX microscope equipped with fluorescent optics and Image Pro Premier software (http://www.mediacy.com/imagepro).