Bp1600 100

Cells were plated onto glass coverslips in 24-well plates and infected at the indicated MOIs. Cells were washed twice with cytoskeletal (CSK) buffer (10 mM HEPES, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 5 mM EGTA), then simultaneously fixed and permeabilized in 1.8% formaldehyde (methanol-free, Thermo Fisher Scientific 28906) and 0.5% Triton-X100 in CSK for 10 minutes. Cells were washed three times in PBS and twice in CSK post-fixation. Coverslips were blocked with 3% bovine serum albumin (BSA, Fisher Bioreagents BP1600-100) prior to Click-chemistry followed by immunostaining. EdC-labeled HSV was detected using the Click-iT Plus EdU Alexa Fluor 555 Imaging Kit (Thermo Fisher Scientific C10638) according to the manufacturer’s instructions, using a working stock of picoyl azide-Alexa Fluor 555 (PCA-AF 555). For immunostaining, samples were incubated overnight with primary antibodies in 3% BSA and washed in PBS three times. Following primary antibody treatment, coverslips were incubated for 1 hour in Alexa Fluor-conjugated secondary antibodies (Invitrogen A-11008). Nuclei were stained with Hoechst 33258 (Life Technologies H3570). Antibodies and their applications are listed in Table S1.

Protein lysates were generated using RIPA buffer (Santa Cruz Biotechnology, catalog 24948A) for 20 minutes on ice, followed by 15 minutes of centrifugation at 13,000 rpm, at 4°C. Protein-containing supernatant was transferred to microcentrifuge tubes and stored at –80°C until further use. Protein was quantified using Bradford Assay (BioRad, catalog 5000006) following manufacturer’s recommendations; standard curves were generated with bovine serum albumin (Fisher, catalog BP 1600-100).

Mouse embryos were dissected in phosphate-buffered saline, pH 7.4 (PBS), fixed in 3.7% formaldehyde overnight at 4°C, and washed in PBS containing 0.1% Triton (PT). Embryos were stored at −30°C in methanol and were rehydrated at the time of use in PT. Embryos were cryopreserved using a sucrose gradient of 10%, 20% then 30% w/v sucrose in PT, followed by 1:1 30% sucrose:OCT Compound (Fisher Scientific 23730571) and 100% OCT. Embryos were flash-frozen in OCT for cryosectioning using dry ice and 100% ethanol bath and were stored at −80°C until sectioning. Cryo-sectioning was performed at 10 um and slides were stored at −80°C until staining. Sections on slides were washed with PT and blocking was performed at room temperature for 1 h in 5% Gibco normal goat serum (16210064) and 1% bovine serum albumin (Fisher Scientific BP1600100). Primary antibodies were diluted in blocking solution and applied to tissue overnight at 4°C [Sox9 (EMD Millipore AB5535; 1:1,000) Pax3 (DSHB PAX3; 1:100)]. Secondary antibodies (AlexaFluor) diluted in blocking buffer (1:500) were applied for 1.5 h at room temperature. Sections were mounted with Fluoromount G (Fisher Scientific OB10001). Images of the cross-sections were taken on Zeiss LSM780 or LSM 980. Wholemount embryos were imaged on Leica M165FC dissecting microscope with a Leica DFC 3000G camera or Zeiss LSM780.