Ab178400

After differentiation into sensory neurons on glass coverslips, cells were washed with PBS and fixed in 4% PFA for 15 min. After washing and permeabilization with 0.2% Triton X-100 for 1 h, coverslips were blocked with 10% BSA for 1 h at RT before incubation with primary antibodies at 4°C overnight. The following primary antibodies were used: Sox10 (#89356, Cell Signalling) at 1:1000 dilution; NEUROG1 (#mab3524, R&D) at 1:500 dilution, BRN3A.
(mab1585, Milipore) at 1:200, ISL1 (AB178400, Abcam) at 1:100 dilution, Tuj1 (MAB1637, Millipore) at 1:500 dilution, PRPH (AB4666, Abcam) at 1:100. After washing, coverslips were exposed to the required Alexa Fluor secondary antibodies (either Thermo Fisher Scientific or Abcam) and were incubated for 1 h at room temperature in the dark. Cultures were counterstained with DAPI and mounted onto slides using Dako mounting medium (Agilent). Images were captured using an EVOS FL Auto2 imaging system (Thermo Fisher) and processed with Fiji.⁴⁵ (link)

Western blot analysis was conducted according to our previous protocol.24 The following antibodies were used: OCT4 (1:1000; Abcam, ab19857), β‐actin (1:2000; CST, #3700), DNMT1 (1:1000; Abcam, ab13537), Ras (1:1000; Abcam, ab52939), Raf1 (1:1000; Abcam, ab137435), P‐ERK (1:1000; CST, #4377s), ERK (1:1000; CST, #4695s), ER alpha (1:1000; CST, #8644s) and ISL‐1 (1:100; Abcam, ab178400).

Cells were washed twice with DPBS (BI, China), and digested with Typsin (BI) for 1 min, folowed by wash with DPBS containing 0.5% BSA (0.5% PBSA). Next, cells were centrifuged and washed once with 0.5% PBSA. The resuspended cells in 200 μl 0.5% PBSA were passed the flow tube to obtain single cells. After that, cells were incubated with the antibodies for 30 min at room temperature. The antibodies used are Anti-NFATc1 (Abcam, ab2796); Anti-ISL1 (Abcam, ab178400); Anti-VE-cadherin (Abcam, ab33168), Anti-CD31 (R&D, FAB35679), Anti-KDR (R&D, FAB3579), Anti-CDH5 (BD Horizon, 561569), Anti-SOX9 (Proteintech, 67439-1-Ig, 1: 200), Anti-HEY1 (Proteintech, 19929-1-AP, 1: 200), Anti-JAG1 (Proteintech, 668909-1-Ig, 1: 200) or (Invitrogen, PA5-86057), Anti-NOTCH1 (Proteintech, 20687-1-AP, 1: 200), Anti-NOTCH4 (Abcam, ab225329, 1:100), Anti-P-SELECTIN (Proteintech, 60322-1-Ig, 1:200) and the isotype control antibodies: mouse IgG (R&D, C002P) and rabbit IgG (Invitrogen, 10500C). After three times washing, cells were treated with secondary antibodies at room temperature for 15 min. Finally, cells were washed twice and resuspended with 300–500 μl 0.5% PBSA, and then analyzed by Accuri C6 flow cytometer (BD Biosciences, USA).