Laminin b

Immunofluorescence was performed on frozen sections. Intestinal tissues were flushed with PBS and fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose, embedded in OCT compound, and sectioned at 12 μm. Antibodies for immunofluorescence are: RHOA (Cell Signaling Technology, #2117, 1:250), cleaved caspase-3 (Cell Signaling, #9661, 1:250), p-histone H3 (Cell Signaling, #9701, 1:250), laminin B (Cell Signaling, #12586, 1:250), E-cadherin (BD Biosciences, #610181, 1:250), β-catenin (BD Biosciences, #610153, 1:250), Na⁺,K⁺-ATPase α (MBL, #D154-3, 1:250), Ki67 (Abcam, #ab15580, 1:200), chromogranin A (Abcam, # ab15160, 1:200), lysozyme (Dako, #A0099, 1:2000), and mucin-2 (Thermo Scientific, #MS-1037, 1:200).
Images were acquired on a Zeiss 710 and Nikon A1R GaAsP Inverted Confocal Microscope. Signal intensity was optimized for control samples and the same settings (laser power/gain/offset) were used for RhoA KO tissue and cells. Phase-contrast images of enteroids were captured on an Olympus IX51 microscope equipped with a Q Color 5 camera and acquired with Q Capture Pro 6.0 software.

National Institutes for Food and Drug Control (Beijing, China) supplied the Saikosaponin A. Antibodies specific for GAPDH, TGFβ, smad2, smad3, smad4, Wnt, β-catenin, GSK3β and laminin B came from Cell Signaling Technology (Beverly, MA). Antibodies including VE-cadherin, CD31, α-SMA and vimentin came from Abcam (Cambridge, CB4 0FL, UK). SRI-011381 and WAY-262611 were purchased from MechemExpress (Monmouth Junction, NJ 08852, USA).