Monoclonal rat anti cd3 clone cd3 12

Caco-2_BBe monolayers grown on 5 cm² Transwell supports were fixed in 2% paraformaldehyde and immunostained as described previously. Snap-frozen tissues were stored at −80°C, after which 5 μm frozen sections were cut and fixed in 1% paraformaldehyde. EdU was detected using the Alexa Fluor 488 click chemistry detection kit (Invitrogen). Immunostaining used affinity-purified rabbit anti-human MLCK1 (2 μg/ml),^{7 (link)} monoclonal mouse anti-MLCK clone K36 (Sigma-Aldrich, 1 μg/ml)^{7 (link)}, affinity-purified rabbit anti-pMLC (Cell Signaling 3671, 1 μg/ml), monoclonal mouse anti-occludin clone OC-3F10 (Invitrogen, 1 μg/ml), monoclonal mouse anti-E-cadherin clone M168 (Abcam, 2 μg/ml), or monoclonal rat anti-CD3 clone CD3–12 (Abcam, 2 μg/ml). Primary antibodies were followed by Alexa Fluor 488- or Alexa Fluor 594-conjugated affinity-purified secondary antibodies (Invitrogen) along with Alexa Fluor 594-conjugated phalloidin (Invitrogen) and Hoechst 33342 (Invitrogen). Stained sections were mounted in Prolong gold (Invitrogen).