Mar 25, 2024

Public workspaceImmunohistochemistry (IHC) Staining Mouse Brain Sections

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3b7bv4o/v1
  • 1Allen Institute for Neural Dynamics;
  • 2Boston University
Holly Myers
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvo3b7bv4o/v1
Protocol Citation: Naveen Ouellette, daphne.toglia, Holly Myers 2024. Immunohistochemistry (IHC) Staining Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3b7bv4o/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: July 26, 2023
Last Modified: June 18, 2024
Protocol Integer ID: 85540
Keywords: Histology, Immunohistochemistry, IHC, Immunolabeling, Antibody
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.
Materials
SampleSample sections to be stained:
  • 50-100 microns thick
  • Fixed in paraformaldehyde
  • Free floating, stored in PBS or PBS + 0.01% Azide
  • Protected from light

Reagents:

Reagent10xPBSAmbionCatalog #AM9624 ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
ReagentSodium Azide 5%Ricca Chemical CompanyCatalog #71448-16
ReagentMilli-Q water

Optional reagents - determined by stain requested:
  • Note these reagents and stained tissue must be protected from light

ReagentDAPI (4',6-diamidino-2-phenylindole, dihydrochloride)Thermo FisherCatalog #62247
ReagentPrimary Antibody
ReagentSecondary Antibody
ReagentNormal Goat SerumVector LaboratoriesCatalog #S-1000-20
ReagentNormal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ML
ReagentBlock AidInvitrogen - Thermo FisherCatalog #B10710
ReagentUrea powderMerck MilliporeSigma (Sigma-Aldrich)Catalog #U5378-100G


MaterialsProduct number
Serological pipet fillerThermofisher, 9501
48 well plateCostar, 3548
Cell culture insertsNetwell, 734-1589
Serological pipetsFisher Scientific, 13-678-11E
Nutating mixerFisherbrand, 88-861-043
Stir plateMerck MilliporeSigma, Z693510
Manual single channel pipettes: P20, P200, P1000Rainin, 30456871
Manual single channel pipettes: P2Rainin, 17014393
Manual dingle channel pipettes: P5000Rainin, 17011790
Stir barGrainger, 21R590




Recipes - blocking and DAPI solutions should be made depending on experimental need

1L 1xPBS:

Combine the following reagents into a container with a stir bar. A graduated cylinder may be used to measure MilliQ water and a graduated cylinder or serological pipet may be used to measure 10xPBS. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:02:00 or until solution is mixed. Store at TemperatureRoom temperature for 1 month.

ReagentVolume
Milli-Q water900 mL
10xPBS100 mL

1L 1xPBS & Sodium Azide 0.01% :

Combine the following reagents into a container with a stir bar, using a graduated cylinder to measure the 1xPBS and a P5000 pipette to measure the 5% sodium azide. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:02:00 or until solution is mixed. Store at TemperatureRoom temperature or Temperature4 °C for up to 1 year.


ReagentVolume
1xPBS998 mL
0.01% Sodium Azide2 mL

1L 1xPBS & Triton X-100 0.2%:

Measure 1xPBS into a container with a stir bar using a graduated cylinder and slowly pipet Triton X-100 in using a P5000 pipette. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:15:00 to ensure Triton X-100 goes into solution. Store at TemperatureRoom temperature for 1 month.


ReagentVolume
1xPBS998 mL
Triton X-1002 mL

1L 1xPBS & Triton X-100 0.06%:

Measure 1xPBS into a container with a stir bar using a graduated cylinder and slowly pipet Triton X-100 in using a P5000 pipette. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:15:00 to ensure Triton X-100 goes into solution. Store at TemperatureRoom temperature for 1 month.

ReagentVolume
1xPBS994 mL
Triton X-1006 mL



2mL 5mg/mL DAPI solution:

Add Milli-Q water to DAPI powder in 10mg vial using P5000 pipette. Vortex until powder completely mixes into solution. Store at Temperature4 °C and vortex before use.

ReagentVolume
Milli-Q water2 mL
DAPI powder10 mg

200 mL 5% Normal Goat Serum & Triton X-100 0.06% & 4M Urea (NGSTU)

Measure urea on scale and add into a container for mixing NGSTU. Add 10x PBS into the container with a serological pipet, then add normal goat serum, Triton and water to container using manual pipette. Add stir bar to container and mix on stir plate at high speed (300 RPM or higher) for Duration00:15:00 to ensure all reagents fully homogenize. This working solution can be stored at Temperature4 °C and used for up to 1 week. Beyond this time, there would be concern for possible microbial contamination that would affect performance.

*Amount of blocking serum made should be determined by number of sections to be stained.

Reagent Amount
Normal Goat Serum10 mL
Urea48 g
10X PBS20 mL
Triton X-100120 uL
Milli-Q WaterFill to 200 mL


200 mL 5% Normal Donkey Serum & Triton X-100 0.2% in PBS (NDST)

Measure 10X PBS into container with a serological pipet, then add Normal Donkey Serum, Triton, and water to container using manual pipette. Add stir bar to container and mix on stir plate at high speed (300 RPM or higher) for Duration00:15:00 to ensure all reagents fully homogenize. This working solution can be stored at Temperature4 °C and used for up to 1 week. Beyond this time, there would be concern for possible microbial contamination that would affect performance.

*Amount of blocking serum made should be determined by number of sections to be stained.

ReagentAmount
Normal Donkey Serum10 mL
10X PBS20 mL
Triton X-100400 uL
Milli-Q WaterFill to 200 mL


200 mL 5% Normal Goat Serum & Triton X-100 0.06% (NGST):
Measure 10X PBS into container with a serological pipet, then add normal goat serum, Triton, and water to container using manual pipette. Add stir bar to container and mix on stir plate at high speed (300 RPM or higher) for Duration00:15:00 to ensure all reagents fully homogenize. This working solution can be stored at Temperature4 °C and used for up to 1 week. Beyond this time, there would be concern for possible microbial contamination that would affect performance.

*Amount of blocking serum made should be determined by number of sections to be stained.

ReagentAmount
Normal Goat Serum10 mL
10X PBS20 mL
Triton X-100400 uL
Milli-Q WaterFill to 200 mL






Safety warnings
Attention
DAPI is a mutagen and should be handled with care. Wear PPE and dispose into hazardous waste stream. Please consult your immediate supervisor or the EH&S manager/representative if you have questions or concerns.

Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.

Paraformaldehyde is carcinogenic. Wear gloves at all times when handling specimen tissue slices fixed in paraformaldehyde, as the tissue may contain trace amounts of the chemical.
Personal Protective Equipment (PPE) should be used at all times while operating this protocol. If you are unsure what PPE you should be using, see your immediate supervisor.
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.
Setup
Setup
Well plate setup:
Select either a plastic well plate or Netwell cell culture inserts for tissue section staining.

Note
A plastic well plate may be helpful for conducting multiple different stains simultaneously, while a Netwell kit may be efficient for applying the same stain to a large number of tissue sections.

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
Staining
3d 0h 10m
Wash mouse brain sections three times in 1xPBS for Duration00:05:00 at TemperatureRoom temperature on shaker using either a plastic well plate or Netwell kit with mesh inserts.

Note
In this step and all subsequent steps, protect tissue from light to prevent bleaching by wrapping well plate in foil while it is on the shaker.

15m
Blocking:
Select blocking solution. See examples of frequently requested blocking solutions below:


Blocking solutionNotesExample
BlockAidCan be paired with any secondary antibodyExample: BlockAid + chicken anti-GFP (primary antibody) + (goat OR donkey) anti-chicken (secondary antibody)
Normal goat serum with triton (NGST)Must match secondary antibodyExample: NGST (blocking solution) + chicken anti-GFP (primary antibody) + goat anti-chicken (secondary antibody)
Normal donkey serum with triton (NDST)Must match secondary antibodyExample: 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 backgroundExample: 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 DurationOvernight

Section thicknessBlocking duration
50 microns1 hour
100 microns2 hours

Wash sections in chosen blocking solution for chosen blocking duration on shaker at TemperatureRoom 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.

Frequently requested primary antibodiesSuggested dilution
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 Duration72:00:00
Section thicknessPrimary antibody staining duration minimums
50 microns24+ hours
100 microns48+ hours


3d
Remove blocking solution and incubate tissue in chosen primary antibody solution on shaker at Temperature4 °C or TemperatureRoom temperature for chosen staining duration.


Note
If no secondary antibody stain is required, skip to step 7.


Wash sections in new wells containing 1xPBS & Triton X-100 0.2% five or more times for Duration00:05:00 on shaker at TemperatureRoom temperature

25m
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.


Frequently requested secondary antibodiesSuggested dilution
DAPI (5 mg/mL stock concentration, then diluted to working concentration) combine with secondary antibody1:1000 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



Note
If additional DAPI stain is required in combination with secondary antibody, continue with step 6.2. If not, skip to step 6.3.


Vortex Concentration5 mg/mL DAPI solution and dilute it to preferred concentration in secondary antibody solution. 1:5000 is a common dilution for DAPI.


Safety information
DAPI is a mutagen, so wear PPE (gloves, lab coat, safety goggles) and dispose in hazardous waste stream.


Choose a staining duration based on section thickness. Suggestion below are minimums and secondary staining duration may run up to Duration48:00:00 :
Section thicknessSecondary staining duration minimuns
50 microns1+ hour
100 microns2+ hours


2d
Incubate sections in chosen secondary antibody solution for chosen staining duration on shaker at TemperatureRoom temperature

Wash sections in 1xPBS & Triton X-100 0.2% three times for Duration00:05:00 on shaker at TemperatureRoom temperature

25m
Wash sections in 1xPBS two times for Duration00:30:00 on shaker at TemperatureRoom temperature

30m
Store samples in 1xPBS for short term use or 1xPBS & Azide 0.01% for long term use (DurationOvernight or up to several weeks) at Temperature4 °C until ready for mounting.


Safety information
Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.


Note