Immunohistochemistry (IHC) Staining Mouse Brain Sections
Naveen Ouellette, Holly Myers, daphne.toglia
Abstract
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
Setup
Well plate setup:
Select either a plastic well plate or Netwell cell culture inserts for tissue section staining.
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.
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.
Staining
Wash mouse brain sections three times in 1xPBS for 0h 5m 0s at Room temperature on shaker using either a plastic well plate or Netwell kit with mesh inserts.
Blocking:
Select blocking solution. See examples of frequently requested blocking solutions below:
| A | B | C |
|---|---|---|
| BlockAid | Can be paired with any secondary antibody | Example: BlockAid + chicken anti-GFP (primary antibody) + (goat OR donkey) anti-chicken (secondary antibody) |
| Normal goat serum with triton (NGST) | Must match secondary antibody | Example: NGST (blocking solution) + chicken anti-GFP (primary antibody) + goat anti-chicken (secondary antibody) |
| Normal donkey serum with triton (NDST) | Must match secondary antibody | Example: NDST (blocking solution) + chicken anti-GFP (primary antibody) + donkey anti-chicken (secondary antibody) |
| Normal goat serum with triton and urea (NGSTU) | Must match secondary antibody, urea helps with background | Example: NDSTU (blocking solution) + chicken anti-GFP (primary antibody) + donkey anti-chicken (secondary antibody) |
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
| A | B |
|---|---|
| 50 microns | 1 hour |
| 100 microns | 2 hours |
Wash sections in chosen blocking solution for chosen blocking duration on shaker at Room temperature
Primary staining:
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.
| A | B |
|---|---|
| Immunostar mouse anti-Tyrosine hydroxylase (22941) | 1:1000 |
| NovusBio rabbit anti-Tph2 antibody (NB100-74555) | 1:1000 |
| Rockland rabbit anti-RFP antibody pre-adsorbed (600-401-379) | 1:1000 or 1:800 |
| Invitrogen mouse anti-HA (26183) | 1:500 |
| AVES chicken anti-GFP (GFP-1020) | 1:800 |
| Immunostar goat anti-5HT (26183) | 1:800 |
| FujiFilm Wako rabbit anti-IBA1 (019-19741) | 1:1000 |
| Sigma mouse anti-GFAP (G6171) | 1:1000 |
Chose a staining duration based on section thickness. Note that the durations below are minimums and sections may be stained up to 72h 0m 0s
| A | B |
|---|---|
| 50 microns | 24+ hours |
| 100 microns | 48+ hours |
Remove blocking solution and incubate tissue in chosen primary antibody solution on shaker at 4°C or Room temperature for chosen staining duration.
Wash sections in new wells containing 1xPBS & Triton X-100 0.2% five or more times for 0h 5m 0s on shaker at Room temperature
Secondary staining:
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.
| A | B |
|---|---|
| DAPI (5 mg/mL stock concentration, then diluted to working concentration) combine with secondary antibody | 1:1000 or 1:5000 |
| Invitrogen goat anti-mouse 488 cross-adsorbed (A11001) | 1:500 |
| Invitrogen goat-anti-rabbit 488 (A11012) | 1:500 |
| Invitrogen goat anti-mouse 647 (A-21236) | 1:500 |
| Invitrogen goat anti-rabbit 405 (A-31556) | 1:500 |
| Invitrogen goat anti-rabbit 647 (A21244) | 1:500 |
| Jackson Immuno goat anti-chicken 488 (703-545-155) | 1:500 |
| Invitrogen goat anti-rabbit 594 (A-11012) | 1:500 |
| Invitrogen donkey anti-goat 647 (A-21447) | 1:500 |
Vortex 5mg/mL DAPI solution and dilute it to preferred concentration in secondary antibody solution. 1:5000 is a common dilution for DAPI.
Choose a staining duration based on section thickness. Suggestion below are minimums and secondary staining duration may run up to 48h 0m 0s :
| A | B |
|---|---|
| 50 microns | 1+ hour |
| 100 microns | 2+ hours |
Incubate sections in chosen secondary antibody solution for chosen staining duration on shaker at Room temperature
Wash sections in 1xPBS & Triton X-100 0.2% three times for 0h 5m 0s on shaker at Room temperature
Wash sections in 1xPBS two times for 0h 30m 0s on shaker at Room temperature
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.
