Feb 20, 2025
Immunohistochemistry for Tyrosine Hydroxylase (TH) Detection in Brain Sections
- Marta Graziano1,2,
- Ioannis Mantas1,2,
- Konstantinos Meletis1,2
- 1Karolinska Institutet;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Marta Graziano, Ioannis Mantas, Konstantinos Meletis 2025. Immunohistochemistry for Tyrosine Hydroxylase (TH) Detection in Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr9jwpvmk/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: February 20, 2025
Last Modified: March 26, 2025
Protocol Integer ID: 123082
Abstract
This protocol describes the immunohistochemical staining procedure for detecting Tyrosine Hydroxylase (TH) in brain sections using primary antibodies and fluorescent-conjugated secondary antibodies. The protocol ensures optimal signal specificity and preservation of tissue morphology.
Materials
Solutions and Buffers:
- 4% Paraformaldehyde (PFA) in PBS (for washing)
- Blocking Buffer: 10% donkey serum, 0.3% Triton-X in PBS
- Primary Antibody Dilution Buffer: 2.5% donkey serum, 0.3% Triton-X in PBS
- Phosphate-Buffered Saline (PBS)
- Mowiol Mounting Medium
Primary Antibodies:
- Chicken anti-TH (Cat# ab76442, Abcam)
Secondary Antibodies:
- Fluorescent-conjugated secondary antibodies against chicken IgG
Equipment:
- Shaker or rotator
- Leica DM6000 B microscope (Leica Microsystems)
- Coated slides and coverslips
- Pipettes and tips
- Humidity chamber (optional for incubation steps)
Section Washing
Section Washing
Wash brain sections once with 4% PFA in PBS.
Ensure complete removal of fixative by gentle shaking or rotating for 5 minutes.
Rinse briefly with PBS to remove any residual PFA.
Blocking Step
Blocking Step
Prepare the blocking buffer (10% donkey serum, 0.3% Triton-X in PBS).
Incubate sections in the blocking buffer for 1 hour at room temperature (RT) to minimize nonspecific binding.
Place the sections on a shaker or rotator to ensure even exposure to the blocking solution.
Primary Antibody Incubation
Primary Antibody Incubation
Prepare the primary antibody solution using the following:
Chicken anti-TH (Cat# ab76442, Abcam)
Dilute 1:200 in 2.5% donkey serum and 0.3% Triton-X in PBS.
Incubate sections with the primary antibodies for 1 hour at RT.
Optional: Incubation can be performed in a humidity chamber to prevent evaporation.
Washing After Primary Antibody Incubation
Washing After Primary Antibody Incubation
Wash the sections three times with PBS for 5 minutes each.
Ensure gentle shaking or rotation during washing steps.
Secondary Antibody Incubation
Secondary Antibody Incubation
Incubate sections with fluorescent-conjugated secondary antibodies specific to chicken IgG dilution 1:1000 for 1 hour at RT.
Protect from light to preserve fluorescence.
Final Washing
Final Washing
Wash sections three times with PBS for 5 minutes each.
Perform the washes in the dark to maintain fluorescence integrity.
Mounting
Mounting
Mount the sections on coated slides.
Cover the sections with coverslips using Mowiol mounting medium.
Allow the slides to dry in the dark for at least 30 minutes before imaging.
Imaging
Imaging
Visualize the sections using a Leica DM6000 B microscope with a 10× objective.
Use appropriate fluorescence filter sets to detect the secondary antibody signals.
Adjust exposure settings to optimize signal-to-noise ratio.