Immunocytochemistry for CASR in iPSc-derived dopaminergic neurons
Peter Kilfeather
Abstract
CASR immunocytochemistry protocol to accompany Kilfeather, Khoo et al., 2023: Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease
Steps
Day 1
Fix the iPSC derived neurons in 4% PFA.
Permeabilise cells for 10 min in a solution of 5% NDS, 1% BSA and 0.5% Triton-X 100.
Block in a solution of 5% NDS and 1% BSA for 1 h.
Incubate cells for 16 h O/N at 4°C with the following primary antibodies: CASR (Abcam Cat# ab79038, RRID:AB_2071489, 1:500), MAP2 (Abcam Cat# ab92434, RRID:AB_2138147, 1:1000), tyrosine hydroxylase (Millipore Cat# AB1542, RRID:AB_90755, 1:500).
Day 2
Wash cells twice in PBS and then incubate in secondary antibodies and DAPI for 1.5 h at RT.
Wash cells 3 times and store in fresh PBS till imaging.
