Donkey anti rat 488

In this study, we used the following antibodies (also specified in relevant Methods sections above): Monoclonal antibodies: rat anti-HA (Roche 3F10, Cat.: 11867423001, Lot: 47877600), mouse anti-FLAG (Sigma M2, Cat.: F1804, Lot: SZCD3524), or mouse monoclonal antibodies produced in-house: anti-HA (12CA5), anti-FLAG 9H1. Polyclonal antibodies against RhopH2, RhopH3 and Clag3 were generated in rabbits by GenScript against the following antigens: RhopH3 T731-Y829, RhopH2 L20-S1378 and Clag3 K1277-H1417 and verified using indirect ELISA by the manufacturer. Rabbit anti-RON4 serum was used as published before^{34 (link)}. The following secondary Alexa 488/594 fluorophores from Life Technologies were used: chicken anti-mouse 594 (Cat.: A21201, Lot: 42099 A), donkey anti-rat 488 (Cat.: A21208, Lot: 2310102), chicken anti-rabbit 594 (Cat.: A21442, Lot: 2110863). The following secondary HRP-conjugated antibodies were used: goat a-rat (Southern Biotech, Cat.: 3030-05, Lot: G2512-M748B), goat α-mouse (Merck Millipore, Cat: AP124P), goat a-rabbit (Merck Millipore, Cat: AP187P).

Anti-α-actinin-4 was from Santa Cruz Biotechnology, Inc (Dallas, TX, USA, Cat #: SC-49333); anti-CD11b was from CedarLane Laboratories Limited (Hornby, Ontario, Canada, Cat #: CL8941AP); anti-Fibronectin was from Sigma (St. Louis, MO, USA, Cat #: F3648); anti-Integrin α8 was from R&D Systems (Minneapolis, MN, USA, Cat #: AF4076); anti-Laminin α1 was a gift from Dr. Dale Abrahamson (KU Medical Center, Kansas City, KS, rat monoclonal 8B3); anti-Laminin α2 and anti-β actin were from Sigma (St. Louis, MO, USA, Cat #: L0663); anti-Laminin α5 was a gift from Dr. Jeff Miner (Washington University, St. Louis, MO); anti-p-FAK³⁹⁷ was from Assay Biotechnology (Sunnyvale, CA, USA, Cat #: A0925) and from Invitrogen (Carlslab, CA); anti-Total FAK was from Cell Signaling Technology (Danvers, MA, USA, Cat #: 3285). Anti-MMP-10 antibodies were from Millipore (Billerica, MA, USA, Cat # ABT 289). All Alexa-fluor conjugated secondary antibodies were from Invitrogen (Carlsbad, CA), including donkey anti-rat 488, donkey anti-rabbit 555, goat anti-rat 488, goat anti-rabbit 555, donkey anti-rabbit 488, and donkey anti-goat 568. The small molecular inhibitor for FAK activation, TAE226 was from Chem Scene (Monmouth Junction, NJ, Cat #CS-0594); the peptide inhibitor for NF-kappaB (SN-50) was from Calbiochem (now EMD Millipore, Billerica, MA, Cat #481480)

FFPE 4 µm thick sections were dewaxed and rehydrated before heat mediated antigen retrieval for 15 min in either TrisEDTA (Ph9), then permeabilised in PBS 0.1% Tween 20 (PBST). Sections were blocked for 30 min with Protein Block (Spring Bio), then incubated with the primary antibodies HNF4α (1:200, Perseus Proteomics, PP-H1415-00), Ly6G (1:500, Biolegend, 127,602) and minichromosome maintenance complex component 2 (MCM2) (1:200, Cell Signalling, 4007S) overnight at 4 °C. Following washing secondary antibodies; Donkey, anti-goat 555, (Invitrogen a32816), donkey anti-rabbit 647 (Invitrogen A32795) and donkey anti-rat 488 (Invitrogen A21208) were applied for 1 h, with DAPI (1:1000) and mounted with flouromount (Southern Biotech).

Cultured cells were fixed by immersion in 4% (w/v) PFA in PBS overnight at 4 °C and PBS washed before primary antibodies were applied in blocking solution (10% FCS/0.2% Triton X-100 in PBS) overnight at 4 °C. After PBS washing, Alexa Fluor-conjugated secondary antibodies, diluted in blocking solution, were applied overnight at 4 °C. Coverslips were washed thrice and mounted with DAKO fluorescent mounting medium (Sigma). Primary antibodies included: rabbit anti-Pcdh15^{32 (link)} [0.45 mg/ml; 1:200; Figs. 1 and 2], rabbit-anti-pan Pcdh15 (HL5614^{29 (link)}, 1:100; Fig. 8), rabbit anti-CD3 Pcdh15 (PB375^{29 (link)}, 1:100; Fig. 8), rat anti-GFP (Nacalai Tesque, 1:2000), goat-anti-PDGFRa (R&D System; 1:100), mouse-anti-Arp2 (Santa Cruz Biotechnology SC376698; 1:10) and mouse-anti-Arp3 (Santa Cruz Biotechnology SC48344; 1:10). Secondary antibodies included: donkey-anti-rat 488 (Invitrogen; 1:1000), donkey-anti-rabbit 568 (Invitrogen; 1:1000), donkey-anti-rabbit 647 (Invitrogen; 1:1000), donkey-anti-rabbit 488 (Invitrogen; 1:1000), donkey-anti-mouse 647 (Invitrogen; 1:1000) and donkey anti‐goat 647 (Invitrogen; 1:1000). Filamentous (F)-actin was also detected by exposing cells to Alexafluor-568 conjugated phalloidin (Invitrogen; 1:50) and cell nuclei were detected by exposing cells to Hoechst 33342 (Thermo Fisher Scientific; 1:1000).

Pancreata were fixed with 4% PFA at 4°C for 3 hr, embedded in 30% sucrose, and frozen in OCT (Tissue-Tek). Pancreatic sections (10 µm) were stained using a standard protocol. The following primary antibodies and dilutions were used: guinea pig anti-insulin (1:6, Dako, IR00261-2), mouse anti-glucagon (1:500, Sigma G2654), rabbit anti-glucagon (1:200, Cell Signaling 2760S), rabbit anti-somatostatin (1:1000, Phoenix G-060-03), rabbit anti-Cx36 (1:80, Invitrogen 36-4600), rabbit anti-Col IV (1:300, Abcam Ab656), rat anti-laminin β1 (1:500, Invitrogen MA5-14657), and rabbit anti-Tubb3 (1:4000, BioLegend poly18020). The following secondary antibodies were used at 1:500: donkey anti-guinea pig 594 (Jackson), donkey anti-guinea Pig 647 (Jackson), donkey anti-rabbit 488 (Invitrogen), donkey anti-rabbit 594 (Invitrogen), donkey anti-goat 647 (Invitrogen), donkey anti-rat 488 (Invitrogen), donkey anti-mouse 488 (Jackson), and donkey anti-biotin 488 (Jackson). Slides were imaged using a Leica SP8 Scanning Confocal microscope or a Zeiss Axio Observer.Z1 microscope.