Jul 17, 2024
Striatal sections immunofluorescence and analysis
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen 2024. Striatal sections immunofluorescence and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnpmxgk5/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: July 17, 2024
Last Modified: July 17, 2024
Protocol Integer ID: 103539
Keywords: ASAPCRN, phosphorylation of ribosomal protein s6, ribosomal protein s6, haloperidol activation, lrrk2 inhibition, phosphorylation, phosphorylation event, mediated phosphorylation event, striatal sections immunofluorescence, haloperidol, canonical pathway, d2r antagonism, pka dependent
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
Previous studies have shown that the phosphorylation of ribosomal protein S6 (p-rpS6) at S235/236 residues increases in iSPNs in response to haloperidol activation. This phosphorylation is cAMP/PKA dependent, which is the canonical pathway inhibited by D2R antagonism. We therefore explored whether LRRK2 inhibition interferes with this haloperidol-mediated phosphorylation event.
Immunostaining
Drd2-eGFP mice crossed with LRRK2-GS KI and control littermates were treated as indicated and perfused with 50 ml of PBS
Mice were perfused with 4% paraformaldehyde in PBS.
Brains were dehydrated with 30% sucrose in PBS for 48hrs and cut coronally (30 μm) by cryostat
Striatal sections were incubated with 5% goat serum in 0.2% Triton X-100 for 2 hrs
the sections were incubated in the same solution overnight at 4 °C with the primary antibodies anti-GFP (1:1000, Invitrogen) and anti-phospho-S6 Ribosomal protein (Ser236/236) (1:300, Cell Signaling Technology)
Sections were washed with PBS for 5mins at RT
Repeat the wash with fresh PBS
Incubate with the secondary antibodies Alexa Fluor‱ 488 and Alexa Fluor‱ 647 (both 1:600, Invitrogen) for 3 hrs.
Sections were washed with PBS for 5mins at RT
Repeat the wash with fresh PBS
Sections were mounted on ProLong Diamond Antifade Mountant
Confocal imaging
Fluorescence images were obtained with Nikon A1R microscope and were acquired with a 20x objective at 1,024 x1,024 pixel resolution
Stitched images of the whole striatum were automatically acquired with Nikon Element
Image analysis
GFP and phospho-S6 Ribosomal protein signal were measured using Imaris 10.1 software
Surface rendering function was used to segment Drd2-eGFP cells
Background subtraction was enabled
The diameter of the largest sphere was set at 15 μm and automatically thresholded
Smooth surface was set to 1.23 μm
Segments were filtered with an area ranging between 100 μm2 and 6,000 μm2
Mean intensity of phospho-S6 Ribosomal protein within Drd2-eGFP surface above 2 times of average phospho-S6 Ribosomal protein channel mean intensity was considered positive