A21202

Lungs were harvested and fixed as previously described (Frank et al., 2019 (link)). They were then washed, dehydrated, embedded in paraffin, and 6-µm sections were obtained. IHC was performed on E15.5, E17.5 and E18.5 sections. In brief, the sections were deparaffinized and rehydrated followed by antigen retrieval (Reveal Decloaker, MSPP-RV1000M, Biocare Medical). The sections were incubated with 3% H₂O₂ for 15 min to quench endogenous peroxidases, blocked with 5% donkey serum, and, finally, incubated with primary antibody (rabbit anti-VWF, 1:250, F3520, Sigma-Aldrich; goat anti-EMCN, 1:200, AF4666, R&D Systems; mouse anti-ACTA2, 1:500, A5228, Sigma-Aldrich; rabbit anti-RFP, 1:50, 600-401-379, Rockland Immunochemicals) in 0.1% PBST overnight at 4°C. The presence of relevant proteins was visualized using Alexa Fluor secondary antibodies (donkey anti-mouse Alexa Fluor 488, 1:250, A-21202, Sigma-Aldrich; donkey anti-rabbit Alexa Fluor 555, A-31572, Thermo Fisher Scientific; donkey anti-goat Alexa Fluor 647 1:250, A-21447, Thermo Fisher Scientific).

The following antibodies were used for immunofluorescence or western blotting: Primary rabbit polyclonal: anti-actin (Sigma-Aldrich, A5060), anti-Daxx (Upstate, 07–471), anti-ATRX (Santa Cruz, H300), anti-PML (Bethyl Laboratories, A301-167A; Jena Biosciences, ABD-030), anti-Sp100 (GeneTex, GTX131569), anti-Mx1 (Santa Cruz, sc-50509; ProteinTech, 13750-1-AP), anti-ISG15 (ProteinTech, 15981-1-AP), anti-ISG54 (IFIT2, proteinTech, 12604-1-AP), and anti-histone H3 (abcam, ab1791). Primary mouse monoclonal: anti-HIRA (Millipore, 04–1488), anti-ICP0 (11060, [114 (link)]), anti-ICP4 (58s, [115 (link)]), anti-VP5 (DM165, [116 (link)]) anti-UL42 (Z1F11; [117 (link)]), and anti-PML (abcam, ab96051). Primary antibodies were detected using the following secondary antibodies: DyLight-680 or -800 conjugated goat anti-rabbit or -mouse (Thermo; 35568 and SA5-35571), Alexa -488, -555, or -647 conjugated donkey anti-rabbit or -mouse (Invitrogen; A21206, A21202, A31572, A31570, A31573, A31571), or HRP conjugated goat anti-mouse (Sigma-Aldrich, A4416).

The following antibodies were used for immunofluorescence or western blotting: Primary rabbit polyclonal: anti-actin (Sigma-Aldrich, A5060), anti-Daxx (Upstate, 07–471), anti-ATRX (Santa Cruz, H300), anti-PML (Bethyl Laboratories, A301-167A; Jena Biosciences, ABD-030), anti-Sp100 (GeneTex, GTX131569), anti-SUMO2/3 (Abcam, ab22654), anti-Mx1 (Santa Cruz, sc-50509;ProteinTech, 13750-1-AP), anti-ISG15 (ProteinTech, 15981-1-AP), and anti-ISG54 (IFIT2, proteinTech, 12604-1-AP). Primary mouse monoclonal: anti-ICP0 (11060, [92 (link)]), anti-ICP4 (58s, [93 (link)]), anti-VP5 (DM165, [94 (link)]), anti-SUMO2/3 (Abcam, ab81371), anti-PML (abcam, ab96051), anti-IFI16 (abcam, ab55328; Santa Cruz, sc-8023). Primary antibodies were detected using the following secondary antibodies: DyLight-680 or -800 conjugated anti-rabbit or -mouse (Thermo; 35568 and SA5-35571), Alexa -488, -555, or -647 conjugated anti-rabbit, or -mouse (Invitrogen; A21206, A21202, A31572, A31570, A31573, A31571), HRP conjugated anti-mouse (Sigma-Aldrich, A4416).