Alexa fluor 488 594 conjugated goat anti rabbit igg

HepG2 cells (4×10⁴) were seeded onto eight-well Lab-Tek II CC² slides (Nunc) one day before infection. IFA for detection of HEV-infected cells was performed as described [62 (link)]. To examine the subcellular localization of MAVS and IRF3, cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.2% Triton X-100 for 15 minutes. Cells were stained with pre-absorbed ch1313 serum and a rabbit anti-MAVS, mouse anti-PMP70, or rabbit anti-IRF3 for 1 h, and subsequently incubated with Alexa Fluor 488/594-conjugated goat-anti-rabbit IgG, Alexa Fluor 488/594-conjugated goat-anti-mouse IgG, or Alexa Fluor 488-conjugated goat-anti-human IgG (Invitrogen) for 1 h. After adding antifade-4 6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), slides were viewed with a Zeiss LSM 510 confocal microscope with a 63x (NA1.2) apochromatic water objective. Images were acquired using the ZEN 2009 software.

The collected organs were embedded in paraffin, sliced, stained routinely with H&E and examined, as well as photographed using a Zeiss microscope. To quantify the lung metastatic burden, the metastatic nodules per lung were counted. For immunohistofluorescence analysis, the lung tissue sections with a thickness of 5 µm were deparaffinized and then repaired with citrate–EDTA antigen retrieval solution. Afterward, lung tissue sections were blocked using FBS containing 0.03% Triton X-100 and then incubated with primary antibodies containing 0.1% Tween 20 for 24 h at 4°C. After washing, lung tissue sections were incubated with secondary antibodies and DAPI solution. After mounting with a mounting medium, all sections were examined under a fluorescence microscope. Information on primary antibodies (1:400) was as follows: PIN1 (Proteintech, China); E-cadherin and Vimentin (CST, USA); N-cadherin, MMP2, SNAIL and HIF1-α (ImmunoWay, USA). Information on secondary antibodies was as follows: Alexa Fluor 488/594-conjugated goat anti-rabbit IgG (Invitrogen, USA).