Immunofluorescence

2.10.1 Preparation of Mowial

Coverslips were mounted using Mowiol solution. To prepare Mowiol solution 12 g glycerol (Sigma) and 4.8 g Mowiol 4-88 (Calbiochem) were added to 12 mL of H2O and mixed overnight at room temperature. The following day 24

mL of 0.2 M Tris (pH 8.5) was added and the solution mixed at 50°C for at least 45 minutes until dissolved. Aliquots were then stored at -20°C until required.

2.10.2 Staining of Acinar cells

Staining of 3D acinar clusters was optimised for clusters of primary cells within collagen gel and was carried out in the glass-bottomed culture dishes. Cells were initially fixed in 4% PFA for 30 minutes at RT before permeablisiation in 0.5% TritonX100 in PBS for 30 minutes. Samples were then washed in PBS once and Glycine Wash buffer (0.1 M glycine in H2O) three times for 10 minutes

each. Blocking was then carried out in cyst block buffer (Table 2.5) for 1 hour before overnight incubation with primary antibody at specific concentrations (Table 2.6) at 4°C. This was followed by 3 x 20 minute washes of cyst block buffer were used. Following this, incubation with a suitable fluorescent secondary antibodies (Alex FluorTM 488 or 647 conjugate, Life technologies) at 1:200 dilution in cyst block buffer for 3 hours at RT in the dark. Secondary antibodies with emission spectra in green (488) and far red (647) were chosen to permit visualisation of cell tracker. Following this, three more washes in cyst block buffer were carried out, each for 20 minutes. Nuclei were then stained for by incubation in Hoescht 33342 (Life technologies) for 15 minutes at room temperature in the dark before mounting of samples in Mowial. Imaging was carried out on a Zeiss LSM 710 confocal microscope.

Reagent

Concentratio

n Supplier

NaCl 130 mM Sigma

Bovine Serum Albumin (BSA) 1% Sigma

Tritonx100 0.20% Sigma

Tween20 0.05% Sigma

FBS 10% ThermoFisher scientific

2.10.3 Staining of Ductal cells

Immunofluorescence staining of 2D pancreatic ductal epithelial monolayers was carried out following established protocols. Throughout this protocol phosphatase inhibitors; sodium orthovanadate and sodium fluoride were both added to all solutions at 1 mM when staining for phosphorylated proteins to inhibit any residual phosphatase activity. All steps were carried out at RT unless otherwise stated. To begin staining, cells were fixed for 10 minutes in 4% PFA followed by 3 x 5 minute washes in TBS. Cells were then permeabilised in 0.25% TritonX-TBS for 10 minutes before blocking in 3% BSA-TBS for 1-2 hours. Cell were then incubated in primary antibody (Table 2.7) in 3%-TBS overnight at 4oC. Following this samples were washed in TBS for 3x 5 minutes before incubating in secondary antibody (1:200, Life technologies) and phalloidin (1:200, Invitrogen) for 1 hours at room temperature. Samples were washed thrice more for 5 minutes each in TBS before incubation with Hoescht 33342 (Life technologies) for 2 minutes. Following this a final H2O wash was carried out for

5 minutes before mounting in Mowiol. Imaging was carried out on a Zeiss LSM 710 confocal microscope.

Antibody Supplier Concentration Source

anti-EphA2 D7 Millipore 05-480 1:200 Mouse

anti-EphA2 8B6 CST-129275 1:200 Mouse

anti-E-cadherin

BD Transduction

610182 1:500 Mouse

anti-Sox9 Millipore AB5535 1:1000 Rabbit

anti-Amylase Sigma A8273 1:200 Rabbit

anti-phosphorylated Src (Y416) CST-21015 1:50 Rabbit anti-phosphorylated Myosin II (Thr18/Ser19) CST-36745 1:50 Rabbit anti-RFP Creative Diagnostics H83194 1:500 Rabbit

2.11 3D Immunofluorescence Tomography

To visualise interactions of normal and KrasG12D pancreatic cells in vivo in 3D a recently developed technique known as 3D immunofluorescence tomography was utilised (Parfitt et al., 2012). This involves embedding of tissue in butyl- methyl methacrylate (BMMA) plastic and cutting serial sections of 2 µm. Slides can be filled with up to 3 ribbons of serial section with each ribbon up to 20 slices or 40 µm. Immunofluorescence of serial sections on slides and subsequent imaging of each section was carried out. Each image can then be aligned to generate a 3D reconstruction of the tissue.

2.11.1 Butyl-methyl Methacrylate (BMMA) formulation

To formulate BMMA, butyl methacrylate and methyl methacrylate monomer solutions (Polysciences) were mixed 1:4 and the reducing agent dithiothreitol was added to a concentration of 5 mM. The photo-initiator, benzoin ethyl ether (Sigma) was also added (0.3% of total volume). Finally, BMMA solution was degassed with N2 for 30 minutes to remove oxygen which inhibits

polymerisation. BMMA solution was stored at -20°C to prevent heat and ambient light inducing polymerisation.

2.11.2 Sample processing

Following fixation of tissue in 2% PFA in PBS for at least 24 hours, samples were embedded in low melting point 3% agarose (Bioline) to orient tissue appropriately. Tissues were then dehydrated in 50%, 70% then 95% ethanol for 30 minutes each at room temperature before a final incubation in 100% ethanol for a further 30 minutes. Following dehydration, samples were infiltrated with BMMA. Samples were incubated with 25%, 50% and 75% BMMA in ethanol for at least 12 hours each at room temperature. Then tissues were submerged and rotated in 100% BMMA for 24 hours at 4°C. For polymerisation of BMMA each sample was placed in a BEEM gelatin capsule and sealed before being placed under UV light for 20 hours at 4oC.

2.11.3 Serial Sectioning

Serial sections 2 µm thick were cut using a diamond knife (DiATOME) on a Leica EM UC7 Ultramicrotome. Cutting was carried out by Dr Geraint Parfitt, a research fellow at Cardiff University. By addition of an adhesive (Pattex) to the top and bottom of the block to bind each section together forming a ribbon. Ribbons of 12-20 sections were then floated onto a Poly-L-Lysine coated slide (ThermoFisher scientific). Chloroform vapour was used when required to expand the sections ensuring sections lay flat before mounting onto slides.

2.11.4 Immunostaining of Serial Sections

Ribbons of serial sections were incubated in acetone for 10 minutes to remove BMMA before rehydration in 95%, 75%, 50% ethanol and PBS each for 10 minutes. Antigens were unmasked by heating sections in 1X citrate buffer (2.94 g Sodium citrate tribasic dehydrate (Sigma) in 1 L H2O, pH 6) in a pressure

cooker. Citrate buffer was heated in a microwave at 1000 W for 10 minutes before submersing slides in the buffer. The pressure cooker was then heated in a microwave for a further 10 minutes. Slides were then cooled in PBS before blocking of non-specific antibody binding. This was carried out by incubating in 5% normal goat serum in PBS for 30 minutes. Tissues were then incubated in primary antibody at the appropriate concentration (Table 2.5) overnight at 4o_C.

Slides were then washed in PBS for 3 x 5 minutes before incubation in secondary antibody (Life technologies) for 2 hours at room temperature. Sections were then incubated in Hoescht 33342 (1:5000 in PBS) for 5 minutes before mounting in 50:50 Glycerol:PBS.

2.11.5 Three-Dimensional Reconstruction

Individual sections were imaged on a Zeiss LSM 710 confocal microscope. The same position in each section was manually selected, checked for focus and then imaged in each channel. The individual montage images were

then merged in Fiji (Version 2.0) to form a single stack maintaining the sequence of sections. For every image acquisition, at least two channels of information were collected with one representing nuclei. Data sets could then be interleaved and co-aligned using the common nuclei channel. Image alignment and three- dimensional reconstruction was performed with the AlignSlices module in the Amira 5.4 Software package (Visage Imaging) with semi-automated alignment. Reconstructions were then exported as TIFF files for analysis in Fiji and Imaris software packages.