Nov 12, 2024
Tissue preparation and immunohistochemistry-Alcacer et al 2024
- 1Alcala University
Protocol Citation: Cristina Alcacer 2024. Tissue preparation and immunohistochemistry-Alcacer et al 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp98d8vzp/v1
Manuscript citation:
Abnormal hyperactivity of specific striatal ensembles encodes distinct dyskinetic behaviors revealed by high-resolution clustering
Cristina Alcacer, Andreas Klaus, Marcelo Mendonça, Sara F. Abalde, Maria Angela Cenci, Rui M. Costa
bioRxiv 2024.09.06.611664; doi: https://doi.org/10.1101/2024.09.06.611664
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: November 01, 2024
Last Modified: November 12, 2024
Protocol Integer ID: 111385
Keywords: ASAPCRN
Abstract
This protocol describes the tissue preparation and immunohistochemistry protocol to verify the extent of dopamine denervation from 6-OHDA injections and check the transfection efficiency of GCamp6f-GFP vial construct that was injected into mouse brains.
Materials
-Perfusion pump
-4% (w/v) paraformaldehyde in phosphate buffered saline (PBS), pH 7.4
-Storage solution: 30% (w/v) ethylene glycol, 30% (w/v) glycerol, and 0.1 M PBS
-Tris-buffered saline (TBS; 0.10 M Tris and 0.14 M NaCl, pH 7.4)
-GFP Polyclonal Antibody, Alexa Fluor 488, Invitrogen (Molecular Probes) Cat#A-21311)
-Rabbit polyclonal α TH antibody Rabbit anti-TH: Peel Freez Biological, Product code: P40101-150
-Alexa Fluor 594 goat anti-rabbit, Jackson ImmunoResearch Labs Cat#111-585-045
-Polyvinyl alcohol mounting medium (PVA-DABCO, Sigma Aldrich)
-Zeiss Axio Imager M2 fluorescence microscope (for imaging final slices)
Before start
Twenty minutes prior the mouse is given an intraperitoneal injection of vehicle (physiological saline) or L-DOPA (6 mg/kg). The mouse is then rapidly anesthetized with pentobarbital (500 mg/kg intraperitoneal injection).
Perfusion & Dissection
Perfusion & Dissection
Stabilize mouse and open the abdomenal cavity of the anesthetized mouse and expose the heart.
Using forceps, stabilize the heart and insert a blunted needle into the left ventricle.
The needle is connected to tubing for the perfusion pump that contains 4% (w/v) paraformaldehyde in saline phosphate buffer (PBS), pH 7.4.
Make a small incision on the right atrium using fine scissors.
Start the perfusion pump with PFA and perfuse until liquid is clear and not bloody (for a total of 5 min infusing 100mL per minute).
Once perfusion is complete, dissect out the brain.
Brain Slice preparation
Brain Slice preparation
Post-fix the brain overnight in 4% (w/v) paraformaldehyde in saline phosphate buffer (PBS), pH 7.4 and store at 4 °C
Cut brains at 30um sections with a vibratome and store at -20 °C in 30% (v/v) ethylene glycol, 30% (v/v) glycerol, and 0.1 M sodium phosphate buffer until sections are processed.
Immunohistochemistry (IHC)
Immunohistochemistry (IHC)
Rinse free floating sections in Tris-buffered saline (TBS; 0.10 M Tris and 0.14 M NaCl, pH 7.4).
Incubate for 5 minutes in TBS containing 3% H2O2 and 10% methanol.
Rinse in TBS (0.10 M Tris and 0.14 M NaCl, pH 7.4).
Incubate for 15 minutes in TBS containing 0.2% Triton X-100.
Block sections in bovine serum solution (BSA).
Incubate overnight at 4 °C with 1:1000, GFP Polyclonal Antibody, Alexa Fluor 488 and 1:1000 rabbit polyclonal α TH antibody.
Rinse in TBS (0.10 M Tris and 0.14 M NaCl, pH 7.4).
Incubate for 45 minutes with 1:400 secondary antibody goat-anti rabbit Alexa Fluor 594 at room temperature.
Rinse twice in TBS (0.10 M Tris and 0.14 M NaCl, pH 7.4).
Rinse twice in TB (0.10 M Tris, pH 7.4).
Mount sections in a polyvinyl alcohol mounting medium.
Imaging confirmation
Imaging confirmation
Use Zeiss Axio Imager M2 fluorescence microscope to confirm lens placement, viral transduction and dopaminergic denervation.