Evos fl auto 2 system

Paraffin embedded tissues were sectioned at 5–6 μm and OCT tissues were cryosectioned (Thermoscientific, UK) at 8–10 μm thickness between -14 to -30°C. All tissues were haematoxylin and eosin (H&E) stained, gut and liver tissue sections were also stained with Gömöri’s Trichrome using previously described methods [12 (link)]. Prior to immunofluorescence (IF) staining, antigen retrieval (10 mM citrate buffer, pH 6) was performed and the sections were avidin/biotin blocked (Vectorlabs, UK). Briefly, pancreatic insulin and glucagon were stained with rabbit anti-insulin (1/200 dilution: Catalogue number Ab181547 Abcam, UK) and mouse anti-glucagon antibodies (1/200 dilution: Catalogue number Ab10988 Abcam, UK) overnight, followed by goat anti-rabbit IgG Alexafluor 488 (1/400 dilution: Abcam, UK) and goat anti-mouse IgG Alexafluor 647 (1/400 dilution: Catalogue A28181 Invitrogen, UK). IL-17 was detected using goat anti-mouse IL-17 (1/100 dilution: Catalogue number Af-421-na R&D Systems, UK) followed by biotinylated rabbit anti-goat IgG (1/200 dilution: Catalogue number 31732 Invitrogen, UK) with streptavidin Alexafluor 647 (1/200: dilution Catalogue number S21374 Invitrogen, UK). IF sections were mounted with Vectashield with DAPI (Vectorlabs, UK) and images were acquired with an EVOS FL Auto 2 system (Thermofisher, UK).

A ThermoFisher EVOS FL Auto 2 System controlled with ThermoFisher EVOS FL Auto 2 V.2.0.2094.0 software using a 40X magnification, 0.75 NA, fluorite, coverslip-corrected objective (ThermoFisher AMEP4699) acquired images at room temperature using the filters: eGFP EX/EM = 470(22)/510(42) nm, smURFP and MsmURFP EX/EM = 635(18)/692(40) nm, and Cy5.5 EX/EM = 665(40)/794(160) nm. Images were 2048 × 1536 pixels with a resolution of 3.45 µm/pixel. Cells were grown on #0 glass-bottom poly-D-lysine-coated dishes.

SH-SY5Y cells were first fixed with 4% PFA for 15 min. Following three PBS washes, the cells were then blocked in 2% BSA with 0.1% Triton-X diluted in PBS for 30 min. Thereafter, cells were incubated with primary rabbit α-syn antibody (conformation specific antibody that primarily labels the α-syn “filament” or aggregate form of the protein; MJFR-14–6-4–2, ab209538, 1:1000) in 0.1% BSA solution for 1 h and following further PBS washes, they were incubated with secondary goat anti-rabbit Alexa Fluor 488 antibody in 0.1% BSA solution for 30 min. Finally, cells were washed three times in PBS and imaged with ThermoFisher EVOS FL Auto 2 system (at × 40 magnification). The labeled α-syn fibrils were counted using an Image-J software particle counting program. Briefly, images of the same magnification and background were imported into Image-J and scale parameters were set. An identical region of interest was demarcated for each image which included the particles of interest. Next, a set threshold was established and applied to all images in order to minimize background fluorescence and capture positive particles.