TITLE: Immunostaining Mouse Brain Tissue or Neuronal Cultures
AUTHORS: Robert Edwards, Shweta Jain

[DESCRIPTION]
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]
SECTION: Tissue fixation
1. Collection of fixed tissues slices from juvenile or adult mice
SECTION: Tissue fixation
1.2. 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
SECTION: Tissue fixation
1.3. Dissect out brain into 15 mL tube containing cold 4% PFA in PBS
SECTION: Tissue fixation
1.4. Incubate   at 4 °C in 4% PFA in PBS

SECTION: Tissue fixation
1.5. Incubate   at 4 °C in 30% sucrose in PBS
SECTION: Tissue fixation
1.1. Anesthetize mouse, pin onto dissection tray and open chest cavity
SECTION: Tissue fixation
1.6. 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) 
 


SECTION: Tissue fixation
2. Fixation of cultured neurons
SECTION: Tissue fixation
1.7. Once slices are collected, IHC is performed on either free-floating sections (in cold 1X PBS) or slide-mounted sections
SECTION: Tissue fixation
2.1. Aspirate culture media
SECTION: Tissue fixation
2.2. Add solution containing 4% PFA and 4% sucrose in 1X PBS to culture dishes
SECTION: Tissue fixation
2.3. Incubate for 10 min at Room temperature 

SECTION: Tissue staining
3. Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker

SECTION: Tissue fixation
2.4. Aspirate fixing solution and add 1X PBS
SECTION: Tissue staining
4. 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

SECTION: Tissue staining
6. Incubate in primary antibody overnight at 4 °C  

SECTION: Tissue staining
5. 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 
SECTION: Tissue staining
5.1. Dilutions for antibodies used in Jain et al., 2023
MAP2 (Chicken) - 1:2000
TH (Mouse & Rabbit) - 1:1000
All other primary antibodies - 1:500
SECTION: Tissue staining
7. Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker
SECTION: Tissue staining
8. Prepare secondary antibody solution by diluting antibody at 1:1000 in 1X PBS containing 2% NGS and 0.2% Triton X-100 
SECTION: Tissue staining
9. Incubate in secondary antibody for 1 hour at 4 °C  in the dark

SECTION: Tissue staining
10. Wash samples three times for 10 minutes at Room temperature in 1X PBS on a rocker
SECTION: Tissue staining
11. If using free-floating sections, mount slices onto a slide. 

For all sample types, mount coverslips using   

SECTION: Imaging
13. 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

SECTION: Tissue staining
12. Store samples at 4 °C for short-term storage or -20 °C for long-term storage

SECTION: Imaging
14. Analyze images using ImageJ Software
SECTION: Imaging
14.1. 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.
SECTION: Imaging
14.2. 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.