Immunostaining Mouse Brain Tissue or Neuronal Cultures
Robert Edwards, Shweta Jain
Abstract
This protocol describes immunohistochemical and immunoctyochemical methods to analyze protein expression in mouse brain. In the protocol, we describe the fixation of tissue or cultured neurons, the immunostaining procedure, and collecting images for analysis.
Steps
Tissue fixation
Collection of fixed tissues slices from juvenile or adult mice
Anesthetize mouse, pin onto dissection tray and open chest cavity
Using a perfusion pump, pierce needle into left ventricle and sever the right atrium; immediately begin perfusing cold 1X PBS followed by 4% paraformaldehyde (PFA) in PBS
Dissect out brain into 15 mL tube containing cold 4% PFA in PBS
Incubate at 4°C in 4% PFA in PBS
Incubate at 4°C in 30% sucrose in PBS
Tissue can be kept in long-term storage at -80°C ; when ready for IHC, slice 35 μm sections using a cryostat (Leica CM3050 S)
Equipment
| Value | Label |
|---|---|
| Cryostat | NAME |
| Leica | BRAND |
| Cryostat | SKU |
Once slices are collected, IHC is performed on either free-floating sections (in cold 1X PBS) or slide-mounted sections
Fixation of cultured neurons
Aspirate culture media
Add solution containing 4% PFA and 4% sucrose in 1X PBS to culture dishes
Incubate for 0h 10m 0s at Room temperature
Aspirate fixing solution and add 1X PBS
Tissue staining
Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker
Block and permeabilize for 2 hours at Room temperature in 1X PBS containing 4% normal goat serum (NGS) and 0.2% Triton X-100 on a rocker
Prepare primary antibody solution by diluting antibody at desired concentration (see step 5.1) in 1X PBS containing 2% NGS and 0.2% Triton X-100
Dilutions for antibodies used in Jain et al., 2023
- MAP2 (Chicken) - 1:2000
- TH (Mouse & Rabbit) - 1:1000
- All other primary antibodies - 1:500
Incubate in primary antibody overnight at 4°C
Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker
Prepare secondary antibody solution by diluting antibody at 1:1000 in 1X PBS containing 2% NGS and 0.2% Triton X-100
Incubate in secondary antibody for 1 hour at 4°C in the dark
Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker
If using free-floating sections, mount slices onto a slide.
For all sample types, mount coverslips using
Store samples at 4°C for short-term storage or -20°C for long-term storage
Imaging
Collect images of fixed tissue using a microscope:
- For high resolution (e.g. to trace axon sections): Nikon Ti Microscope, equipped with CSU-W1 spinning disk confocal, Plan Apo VC 100x/1.4 oil or 60x/1.4 oil objective and Andor Zyla sCMOS camera
- For whole brain sections: Nikon 6D conventional wide-field microscope, Plan Apo 20x/0.75 air objective and DS-Qi2 monochrome camera
Analyze images using ImageJ Software
In Jain et al. (2023), measurement of brain slices involved tracing one-pixel lines of dendrites and representative sections of axons. Fluorescence intensity of VMAT2-HA and TH per μm process length from 20 processes was used to determine the mean intensity per field.
In Jain et al. (2023), measurement of cultured neurons involved tracing one-pixel lines of dendrites and axons. Mean intensity per µm process length was determined in either three segments of the same axon or in three separate dendrites of the same neuron.
