Alexa flour 488 goat anti rabbit igg h l

Mouse eyeballs were fixed in 4% PFA at 4 °C overnight. To prepare frozen sections, the eyeballs were immersed in sucrose and embedded in an OCT compound. Immunofluorescence staining was performed using primary antibodies. Sections were incubated with Alexa Flour 574 goat anti-mouse IgG (H + L) (A11005; Invitrogen, Thermo Fisher Scientific) or Alexa Flour 488 goat anti-rabbit IgG (H + L) (A11008; Invitrogen) for 1 h without light at room temperature. Then sections were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio Biotechnology, Beijing, China) for 5 min. Images were captured using a fluorescence microscope (DM5000B; Leica, Weltzar, Germany). ImageJ image analysis software was used to quantify relative fluorescence intensities in the images.

The primary antibodies used in this study were specific for G3BP1 (Abcam; catalog no.: b181150), G3BP1 (Proteintech; catalog no.: 13057-2-AP), TIAR (Cell Signaling Technology; catalog no.: 8509S), GFP (Abcam; catalog no.: ab290), FLAG (Cell Signaling Technology; catalog no.: 14793S), HA (Cell Signaling Technology; catalog no.: 3724S), GAPDH (Proteintech; catalog no.: 10494-1-AP), ASFV p30, p72 (prepared in our laboratory), and pS273R (gifted by Professor Jianzhong Zhu). IRDye 800CW goat antimouse immunoglobulin G (IgG) (H + L) (catalog no.: 926-32210) was purchased from Sera Care, and IRDye 800CW goat anti-rabbit IgG (H + L) (catalog no.: 925-32211) was purchased from LI-COR. Alexa Flour 488 goat anti-rabbit IgG (H + L) and Alexa Flour 594 goat antimouse IgG (H + L) were all purchased from Invitrogen. The protein agarose A/G used for co-IP was purchased from Santa Cruz Biotechnology (catalog no.: 20397). Sodium arsenate (Sigma–Aldrich; catalog no.: S7400) was dissolved in water at the storage concentration of 500 mM.

The tumor tissues were fixed in paraformaldehyde (4%) at room temperature for 48 h and then embedded in paraffin. Sections of 5 µm in thickness were cut for further analyses. The sections were deparaffinized, rehydrated, antigen retrieved and subsequently blocked with blocking buffer (10% goat serum, 0.5% Triton X-100 in 1×PBS). For IHC, the slides were first incubated with primary antibodies against indicated primary antibodies at 4 °C overnight, and then incubated with HRP-conjugated anti-rabbit antibody at room temperature for 1 h. Then, the sections were incubated with DAB and further counterstained with hematoxylin. For IF, the slides were first incubated with primary antibodies against CD31 at 4 °C overnight and then incubated with Alexa Flour 488 goat anti-rabbit IgG (H + L) (1:500, A11008, Invitrogen, Waltham, MA, USA) and DAPI (1 μg/mL, D9542, Sigma, Livonia, MI, USA) at room temperature for 2 h. Images were taken by using a microscope (Nikon, Tokyo, Japan), and the staining intensity was quantified with Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Details of all antibodies are listed in Table S2.