Rat anti e cadherin

The primary antibodies used in this study are as follows: rabbit anti-Lyz (1:800; Dako, #A0099), rabbit anti-Olfm4 (1:500; Cell Signaling Technology, #39141), recombinant rabbit anti-aldolase B + aldolase C (1:300; Abcam, #ab75751), mouse anti-human KRT20 (1:500; Dako, #M701929-2), mouse anti–Chr-A (1:50; Santa Cruz Biotechnology, #sc-393941), rat anti–E-cadherin (1:400; Santa Cruz Biotechnology, #sc-59778), CD24 Monoclonal Antibody (1:200; Thermo Fisher Scientific, #17-0242-80), and mouse anti-Ki67 (1:200; BD Biosciences, 550609). The secondary antibodies used in this study are as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175654), Goat Anti-Rat IgG H&L (Alexa Fluor 555) preadsorbed (1:1000; Abcam, #ab150166), Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) (1:500; Thermo Fisher Scientific, #A31571), and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 405) preadsorbed (1:1000; Abcam, #ab175649). The dyes used in this study are as follows: WGA conjugated to CF488A (5 μg/ml; Biotium), RedDot1 Far-Red Nuclear stain (1:200; Biotium), and SYTOX Orange Nucleic Acid Stain (1:5000; Thermo Fisher Scientific, #S11368). The order of staining, optimized to ensure good staining quality for all cell types, was based on the staining quality and stripping difficulty of each antibody (fig. S1G).

Cells were grown on coverslips and fixed with 4% PFA in phosphate buffer pH 7.4 for 7 min or with methanol at −20 °C for 10 min. After washing three times with PBS cells were incubated for 1 h with PBS + 2.5% horse serum and 0.05% Saponin (PBSS) or 0.1% Triton X-100 (PBST). Subsequently, primary antibodies diluted in the same solution were added for 2 h at RT or overnight at 4 °C. After washing three times with PBST/PBSS the coverslips were incubated with the secondary antibody (diluted 1:1000 in PBSS + HS/PBST + HS), DAPI (1:1000, Invitrogen Life Technologies) for 1 h. Finally, coverslips were washed with PBS and mounted in mowiol. The following primary antibodies were used: mouse anti-Pals1 (1:100, Santa Cruz #365411), rabbit anti-Pals1 (1:1000, Proteintech #17710-1AP), rabbit anti-PATJ (1:100, raised in this study), mouse anti-PATJ (1:50, DSHB Hybridoma Product AFFN-INADL-1-3G6), rabbit anti-ZO-1 (1:100, Cell Signaling #13663), rabbit anti-E-Cadherin (1:100, Cell Signaling #3195), mouse anti-E-Cadherin (1:100, Santa Cruz #21791), rat anti-E-Cadherin (1:100, Santa Cruz #59778), guinea pig anti-Crb3a (1:200, raised in this study), mouse anti-Occludin (1:100, Santa Cruz #271842).