TITLE: Immunohistochemistry (IHC) Staining Mouse Brain Sections
AUTHORS: Naveen Ouellette, daphne.toglia, Holly Myers

[DESCRIPTION]
The Immunohistochemistry (IHC) Staining for Mouse Brain Sections protocol details the blocking, primary, and secondary antibody staining of 50-100 micron mouse brain tissue slices fixed in 4% PFA. The protocol includes suggested staining duration based on slice thickness for each staining step and tables of the most frequently requested blocking serums and primary and secondary antibody stains and dilutions.

[BEFORE_START]
This protocol details staining of mouse brain tissue that has already been sectioned. Reference the following protocol for instructions on sectioning a whole mouse brain fixed in 4% PFA: Sectioning Mouse Brain with Sliding Microtome

For all steps, protect tissue from light in order to preserve fluorescence by wrapping well plate in foil between steps and during washes and stains.

[STEPS]
SECTION: Staining
None. Wash sections in 1xPBS & 0.01% Azide three times for 5 min in well plate or Netwell cell culture inserts on shaker at Room temperature . 


 


SECTION: Staining
None. Blocking:
SECTION: Staining
None. Choose a blocking solution:
 


SECTION: Staining
None. Chose a blocking duration based on section thickness. Note that the following times are minimums and sections may be blocked up to   
 


SECTION: Staining
None. Incubate sections in chosen blocking solution for chosen blocking duration time on shaker at Room temperature.

SECTION: Staining
None. Primary staining:
SECTION: Staining
None. Chose a primary antibody and dilution based on tissue labeling. Primary antibody should be diluted in blocking serum, with the exception that if BlockAid is used as a blocking serum then the primary antibody should be diluted in 1xPBS. See examples of frequently requested primary antibodies and dilutions below:

 

SECTION: Staining
4.2. Chose a staining duration based on section thickness. Note that the durations below are minimums and sections may be stained up to 4320 min 
 


SECTION: Staining
4.3. Remove blocking solution and incubate tissue in chosen primary antibody solution on shaker at 4 °C or Room temperature for chosen staining duration.


 


SECTION: Staining
8. Wash sections in 1xPBS two times for 30 min on shaker at Room temperature

SECTION: Staining
6. Secondary staining: 
SECTION: Staining
6.1. Choose a secondary antibody and dilution based on the primary antibody used. See table below for commonly requested secondary antibodies and dilutions. If using NDST, NGST, or NGSTU as blocking serum, dilute the secondary antibody in tube with blocking serum. If using Block Aid as blocking serum, dilute the secondary antibody in tube with 1xPBS & Triton X-100 0.2%. Mix secondary antibody dilution mixture in tube by gently swirling.


 


SECTION: Staining
6.2. 
 


Vortex 5 mg/mL DAPI solution and dilute it to preferred concentration in secondary antibody solution. 1:5000 is a common dilution for DAPI.


 

   

SECTION: Staining
6.3. Choose a staining duration based on section thickness. Suggestion below are minimums and secondary staining duration may run up to 2880 min :
 


SECTION: Staining
6.4. Incubate sections in chosen secondary antibody solution for chosen staining duration on shaker at Room temperature 

SECTION: Staining
5. Wash sections in new wells containing 1xPBS & Triton X-100 0.2% five or more times for 5 min on shaker at Room temperature 

SECTION: Staining
9. Store samples in 1xPBS for short term use or 1xPBS & Azide 0.01%  for long term use (  or up to several weeks) at 4 °C until ready for mounting. 


 


 



SECTION: Sectioning
None. Section the tissue:
SECTION: Sectioning
None. Take down:
SECTION: Sectioning
None. Select desired sections for staining:
SECTION: Staining
7. Wash sections in 1xPBS & Triton X-100 0.2% three times for 5 min on shaker at Room temperature 

SECTION: Staining
4. Primary staining:
SECTION: Staining
3. Blocking:
SECTION: Staining
3.1. Select blocking solution. See examples of frequently requested blocking solutions below:


 

SECTION: Staining
3.2. Choose a blocking duration based on section thickness. See table below. Note that suggested blocking durations are minimums and sections may be blocked up to   

 

SECTION: Staining
3.3. Wash sections in chosen blocking solution for chosen blocking duration on shaker at Room temperature 

SECTION: Staining
4.1. Choose a primary antibody and dilution based on tissue labeling. See table below for commonly requested primary antibodies and dilutions. If using NDST, NGST, or NGSTU as blocking serum, dilute the primary antibody in tube with blocking serum. If using Block Aid as blocking serum, dilute the primary antibody in tube with 1xPBS & Triton X-100 0.2%. Mix primary antibody dilution mixture in tube by gently swirling.

 

SECTION: Staining
2. Wash mouse brain sections three times in 1xPBS for 5 min at Room temperature on shaker using either a plastic well plate or Netwell kit with mesh inserts.

 

SECTION: Staining
None. Fill wells with 1xPBS and place tissue sections to be stained into wells.

If there are a large amount of tissue sections, the sections may be double or triple stacked in each well in the well plate (or more if using Netwell insert), but ideally sections in the same well should be separated by at least a few hundred microns (ex: every 6th section or more) so it is easier to see anatomical differences and determine section order when mounting.
SECTION: Setup
1. Well plate setup:
SECTION: Setup
1.1. Select either a plastic well plate or Netwell cell culture inserts for tissue section staining.

 

SECTION: Setup
1.2. Fill wells with 1xPBS and place tissue sections to be stained into wells.

If there are a large amount of tissue sections, the sections may be double or triple stacked in each well in the well plate (or more if using Netwell insert), but ideally sections in the same well should be separated by at least a few hundred microns (ex: every 6th section or more) so it is easier to see anatomical differences and determine section order when mounting.
SECTION: Setup
1.3. If tissues will be stained in different conditions (ex: staining sections with different primary or secondary antibodies) it is best to draw a map of these conditions on the plastic well plate lid and a corresponding map in lab notebook.