Opera phenix plus high content screening system

Cell lines were treated for 48 h with a dose response of A947 prior to pulsing for 15 min with 10uM EdU. Cells were subsequently fixed in 4% paraformaldehyde for 10 min, washed 3 times with PBS and then blocked and permeabilized in PBS containing 10% FBS, 1% BSA, 0.1% TX-100, and 0.01% NaN3 for 1 h at room temperature. Permeabilization buffer was removed and the cells were washed 3× with PBS. The Click-iT® reaction was perform according to the manufacturer’s (Invitrogen C10337) protocol. Following a 30 min incubation in the dark, the cells were washed 3 times with PBS. For nuclear staining, cells were treated with Hoechst 33342 (ThermoFisher) at 1:10000 for 10 min at room temperature. Cells were then washed again 3 times with PBS and imaged on Opera Phenix Plus High-Content Screening System (PerkinElmer). Image analysis was conducted with MATLAB standard and custom-written scripts (https://github.com/scappell/Cell_tracking). For each cell, the integrated nuclear Hoechst signal and EdU positivity were used to determine cell cycle phase and values were averaged for each treatment.

iPSC derived neurons were grown in a black 96 well half area plates with transparent bottom (Greiner) and fixed in the plate with 4% paraformaldehyde (PFA) for 5 min. Cells were permeabilised with 0.05% Saponin for 20 min and blocked for 30 min in PBS containing 10% goat serum at RT, before incubation with primary antibodies overnight at 4°C in PBS containing 0.1% Tween and 1% goat serum. Antibodies used as follows: Map2 (Abcam, #AB92424, 1:1000), Homer1 (Synaptic Systems, #160003, 1:500) and Synapsin I/II (Synaptic Systems, #106004, 1:500). AlexaFluor-conjugated secondary antibodies were incubated for 1 h at RT in PBS containing 0.1% Tween. Nuclear DNA was stained with DAPI (Thermo Fisher Scientific) for 5 min at RT. Images were captured by the Opera Phenix Plus High Content Screening System (Perkin-Elmer).

Immunofluorescence and RNA-FISH sections and transfected cells were imaged using an Operetta high-content imaging system in non-confocal mode using a 20x high-numerical-aperture (0.8) objective (Perkin Elmer Life and Analytical Sciences, Waltham, MA). Immunofluorescence imaging for CTR, CGRP, and amylin was also performed using a 63x high numerical-aperture (1.15) objective with the Opera Phenix Plus High-Content Screening System in confocal mode (Perkin Elmer Life and Analytical Sciences). Image processing and quantification of the immunofluorescence in mouse, rat and human DRG is described in detail in the Supplemental Methods.

Cells were fixed with 4% PFA for 15 min at room temperature. Then blocked and permeabilized in 5% BSA and 0.1% Triton X-100 in PBS for 1 h. Primary antibodies (Supp Table 1) were diluted in 5% BSA in PBS and incubated overnight at 4 °C. The cells were washed 2 times in PBS and incubated in secondary antibodies diluted 1:1000 in 5% BSA in PBS for 1 h. Cells were incubated with DAPI for 10 min and washed 2 times with PBS. Cells were visualised using the Opera Phenix^® Plus High Content Screening System (Perkin Elmer) using the 20 × and 40 × water objective. Image analysis was performed in the Columbus Image Data Storage and Analysis System (Perkin Elmer).
Cryo-sectioned slides were air-dried at 37 °C for 5 min, permeabilized with 0.5% Triton X-100 in PBS for 30 min, and blocked with 5% FBS in PBS for 1 h. Primary antibodies were diluted in the blocking buffer and incubated overnight at 4 °C. After washing with PBS, sections were incubated with a 1:1500 dilution of secondary antibody in the blocking buffer for 1 h at room temperature. DAPI stain was added for 10 min to visualise nuclei. Slides were washed, mounted on coverslips using VECTASHIELD Mounting Medium, and sealed with clear nail polish. Visualisation and analysis were performed using the Nikon Ti-E; Zyla microscope.