Jul 10, 2023
Immunocytochemistry for CASR in iPSc-derived dopaminergic neurons
- 1University of Oxford
Protocol Citation: Peter Kilfeather 2023. Immunocytochemistry for CASR in iPSc-derived dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1onyplr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2022
Last Modified: July 10, 2023
Protocol Integer ID: 71469
Funders Acknowledgement:
ASAP
Medical Research Council
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
Day 1
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
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.