Dec 19, 2024
Immunohistochemistry with DAB
This protocol is a draft, published without a DOI.
- Ingvild Elise Bjerke1
- 1University of Oslo
Protocol Citation: Ingvild Elise Bjerke 2024. Immunohistochemistry with DAB . protocols.io https://protocols.io/view/immunohistochemistry-with-dab-dvfc63iw
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: December 18, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 115908
Keywords: immunohistochemistry, DAB, brain sections, neuroscience, immunohistochemical protocol with dab visualization, immunohistochemical protocol, primary antibody, antibody, secondary antibody, immunohistochemistry, new antibody, dab visualization, testing different dab reaction time, different dab reaction time, dab, mixing nearby section, sections in each well, nearby sections in the same well
Funders Acknowledgements:
EU Horizon Europe
Grant ID: 101147319
Research Council of Norway
Grant ID: 269774
Abstract
This protocol describes an immunohistochemical protocol with DAB visualization. It is tailored for processing sections in 24-well plates, with 1-2 sections in each well. This is sufficient for processing one whole mouse brain with a sampling of one in six sections. This setup gives a high level of control of the order of sections by not mixing nearby sections in the same well. For setups with wells of different size or number, the protocol can be used but different amounts of solutions might be required.
The protocol does not instruct on what antibody to use, but can be tailored for any primary antibody and biotinylated secondary antibody. When tailoring the protocol for a new antibody, it is advisable to run a concentration gradient as well as testing different DAB reaction times.
Protocol materials
Vectastain ABC Kit Rabbit IgGVector LaboratoriesCatalog #PK-6101
DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3-diaminobenzidine)Vector LaboratoriesCatalog #SK-4100
Troubleshooting
Preparations
Prepare wells with sections on the day before or on the morning of Protocol Day 1. Select every sixth section and place each one individually in a wells from anterior to posterior. If the section number in the series exceeds the total number of wells (48), the most posterior ones can be put in the same wells as the most anterior ones, as these will be easy to distinguish when mounting. The last well of the plates should be reserved for a negative control section. Mark the well lid with the section number(s) in each well for reference. It is also advisable to copy down the section numbers processed in each experiment on a paper form for recording purposes. An example of two 24-well plates with section numbers are given below:
| 1 | 2 | 3 | 4 | 5 | |
| A | 003, 285 | 009, 291 | 015, 297 | 021, 303 | 027, 309 |
| B | 039 | 045 | 051 | 057 | 063 |
| C | 075 | 081 | 087 | 093 | 099 |
| D | 111 | 117 | 123 | 129 | 135 |
| 6 | |
| A | 033, 315 |
| B | 069 |
| C | 105 |
| D | 141 |
| 1 | 2 | 3 | 4 | 5 | |
| A | 147 | 153 | 159 | 165 | 171 |
| B | 183 | 189 | 195 | 201 | 207 |
| C | 219 | 225 | 231 | 237 | 243 |
| D | 255 | 261 | 267 | 273 | 279 |
| 6 | |
| A | 177 |
| B | 213 |
| C | 249 |
| D | neg. control |
Prepare the buffer, blocking and incubation solutions needed in Day 1 and 2. The blocking and incubation solutions can be prepared in large batches and kept frozen until use. Using normal serum from the species of the secondary antibody is recommended.
Primary and secondary antibody solutions should be prepared on the day of use.
For primary antibodies, concentrations in the range of 1:1000-1:10,000 are typically used. If mixing from fully concentrated antibody, it is advisable to first make a solution of 1:100 before diluting further. Before pipetting from the concentrated antibody, place the tube in the centrifuge and centrifuge for ~10 seconds to make sure all the liquid is at the bottom of the tube. Add incubation buffer to a tube before adding the concentrated antibody. Always make sure that you see the antibody liquid in the pipette and that it is expelled into the incubation buffer liquid. Pipette up some of the liquid a few times to make sure all the antibody is out of the pipette.
For secondary antibodies, concentrations should generally be higher than that of the primary antibody and typically is in the range of 1:500-1:1000. This concentration can be diluted directly from the concentrated antibody.
Day 1: Blocking and incubation in primary antibody
Wash sections in Phosphate Buffered Saline, 3 x 10 minutes.Room temperature
Block sections with 3% Hydrogen Peroxide for 5 minutes. Room temperature
Safety information
Hydrogen peroxide is a harmful substanse (see Safety Data Sheet). Work in a fume hood and discard of the waste in a glass beaker. When finished, discard of all the waste in a designated waste bottle for H2O2.
.
Do two rows of six wells at a time. Start by pipetting out PBS from the first row, add H2O2 to these six. Start a clock for 5 minutes once you add H2O2 to the first well. Then pipette out PBS from the second row, add H2O2 to these. When the 5 minutes are passed, start by pipetting out H2O2 from the first row, add PBS, and continue with the second row.
Wash sections in Phosphate Buffered Saline, 3 x 10 minutes. Room temperature
Block sections with block buffer (PBS with 10% normal serum, 1% bovine serum albumin and 0.5% triton) for 1 hour. Room temperature
Mix the blocking buffer vigorously immediately before use. Add 300 µl solution to each well. Add a sheet of parafilm under the lid of the well to avoid evaporation of the liquid and incubate for one hour with gentle rotation.
While blocking sections, prepare the primary antibody solution (see Step 1 for general advice). You will need a total of 15 ml finished antibody solution.
Incubate sections in primary antibody overnight (primary antibody in PBS with 3% normal serum, 1% bovine serum albumin and 0.1% triton). 4 °C
Mix the incubation solution vigorously immediately before use. Add 300 µl solution to each well. Add a sheet of parafilm under the lid of the well to avoid evaporation of the liquid and incubate overnight with gentle rotation at 4 degrees Celcius.
Note
NOTE: The last section on the well should be used for negative control, meaning it should be incubated with INCUBATION BUFFER ONLY at this step.
Day 2: Incubation in secondary antibody and mounting
Wash sections in Phosphate Buffered Saline with 0.2% triton, 3 x 10 minutes. Room temperature
While washing sections, prepare the secondary antibody solution (see Step 1 for general advice). You will need a total of 15 ml finished antibody solution.
Incubate sections in secondary antibody for 1 hour. Room temperature
Mix the incubation solution vigorously immediately before use. Add 300 µl solution to each well. Add a sheet of parafilm under the lid of the well to avoid evaporation of the liquid and incubate for one hour with gentle rotation.
Wash sections in Phosphate Buffered Saline with 0.2% triton, 3 x 10 minutes. Room temperature
While washing sections, prepare ABC solution. Vectastain ABC Kit Rabbit IgGVector LaboratoriesCatalog #PK-6101
Add 15 ml PBS to a 50 ml falcon tube. To this, add 6 drops of solution A and 6 drops of solution B. Mix immediately on mixer. The ABC should be allowed 15-30 minutes to react before being used.
Incubate sections in ABC solution for 30 minutes. Room temperature
Add 300 µl solution to each well. Add a sheet of parafilm under the lid of the well to avoid evaporation of the liquid and incubate for 30 minutes with gentle rotation.
Wash sections in Phosphate Buffered Saline with 0.2% triton, 2 x 10 minutes. Room temperature
Wash sections in Phosphate Buffered Saline, 10 minutes. Room temperature
Prepare for DAB staining.
DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3-diaminobenzidine)Vector LaboratoriesCatalog #SK-4100
DAB staining should be performed in a fume hood. Before starting, prepare the following in a fume hood:
- A beaker of sterile water
- An empty beaker for DAB waste
- An empty beaker for PBS waste
- Pipette tips and pipette
- Waste box for dirty pipette tips
- Stop watch
- DAB solution (prepare immidiately before use). Add 15 ml PBS to a 50 ml falcon tube. Add 6 drops of reagent 1, 12 drops of reagent 2, and 6 drops of reagent 3. Mix immidiately.
Incubate sections with DAB kit. Room temperature
The exact time of DAB incubation should be tailored for each antibody individually. It is important that the timing is as precise as possible, so process only a few sections at a time. Depending on the exact length of the reaction time, more sections can be processed. If the reaction is very fast, the DAB solution can be stored on ice to slow it down and thus increase reaction time.
Example procedure for a setup with 3 minutes reaction time, processing 3 wells at a time:
- Remove PBS from all three wells. Add 300 µl DAB to the first well. Press start on a stop watch. At 30 seconds, add DAB to well 2; at 1 minute, add DAB to well 3.
- When 3 minutes have passed, remove DAB from the first well and add 300 µl of sterile water (Step 19). Make sure that there is approximately 30 seconds between removing the DAB from each well.
- When all sections have been changed to sterile water, the timer should be at ~4 minutes minutes (then you know that all the sections have been incubated for the right amount of time).
- Continue with the rest of the well plate, three wells at a time.
- Make sure to change previous rows to the second MQ water wash and PBS (Step 19 and 20) along the way. It is important that the sections are not left for very long in MQ water. Processed sections can be stored in PBS until all sections are done.
Safety information
DAB is a harmful substanse. Work in a fume hood and discard of the waste in a glass beaker. When finished, discard of all the waste in a designated waste bottle for DAB.
Wash sections in sterile water, 2 x 10 minutes. Room temperature
Wash sections in Phosphate Buffered Saline, 10 minutes. Room temperature
Mount sections from PBS onto gelatinized slides.
Label the slide with the animal ID, staining type and section number. Pour buffer into a small petri dish. Insert the bottom part of the glass slide into the buffer. Add the first section to float in the buffer, and use a small paintbrush to slide it onto the slide. To make it slide towards the top of the glass, make a small strip of buffer, it will slide along the liquid. Use a small piece of paper to dry of excess buffer from around the section.
Example of slide label and mounted sections. The slide label reads "P35, ID 111, CALB, 114, 120, 126", indicating the age, ID, and staining of the animal in addition to the section numbers on the slide. Sections should be mounted from left to right to reflect their order and not too close together, to facilitate automatic scanning.
Let the slides dry until all the water is evaporated, to avoid risk the sections sliding off the glass during dehydration. Room temperature
Dehydrate slides in an ascending series of ethanol to xylene.Room temperature
Dehydrate sections in ethanol and clear in xylene. A slide rack staining set will make this process easier. Always make sure there is enough liquid in the rack container to cover the entire glass slide (but not the label part). Place slides in the rack and dehydrate as follows:
- 70 % ethanol, 2 minutes
- 96 % ethanol, 2 minutes
- 96 % ethanol, 2 minutes
- 100% ethanol, 2 minutes
- 100% ethanol, 2 minutes
- Xylene, 5 minutes
- Xylene, 5 minutes
Let the sections stay in xylene while they are coverslipped individually.
Coverslip with Eukitt.Room temperature
Place the glass slide tilted up against a thick pencil. Add a strip of mounting medium (Eukitt) onto the sections. With your left hand, hold a coverslip glass over the slide, lower it carefully from the top towards the bottom of the slide. At the same time, use the end of a paintbrush to push the coverslip glass towards the sections. Be careful not to use too much force, as the coverslip can easily break. Make sure that the mounting medium covers all the sections and that there are no air bubbles on the sections.
Keep slides in the fume hood overnight before scanning, allowing fumes to evaporate.