Protocol for Immunohistochemistry on Mouse Spine
Azadeh Nasrazadani
The Van Den Berg Lab, UT Austin
Abstract: Bone immunohistochemistry is a relatively
tricky procedure. This protocol ensures that mouse spine tissue will
stay on the slide throughout the process.
Procedure
Fixation/Embedding:
- Harvest mouse spine.
- Remove surrounding muscle tissue thoroughly and
carefully.
- Wash tissue in cold PBS then cut spine into 2 or 3
pieces (to later fit easily into a cassette)
- Fix with 4% paraformaldehyde overnight at 4°C.
- Transfer tissue into Decalcification Solution
(Decalcifying Solution, Krajian, J.T.Baker, Cat. # G161-02)
for 4-5 days at 4°C.
- Transfer tissue to a tissue cassette and store in 70%
ethanol until tissue is processed and paraffin embedded.
Sectioning:
- Cut bone sections at 7 micron thickness and place on to
poly-L-Lysine coated slides.
- Place slides on a slide warmer at 55°C for 90 minutes.
- Store slides at room temperature overnight in an upright
position.
De-waxing & Re-hydration:
- Dunk slides in Citrosolv™ for 2 minutes (3X).
- Dunk slides consecutively from 100%, 90%, 80%, and 70%
ethanol for 2 minutes in each solution.
- Re-hydrate tissue in distilled H2O for 2
minutes (2X).
Antigen Retrieval:
- Incubate tissue slides with working
Proteinase K
solution
in a humidified chamber for 7 minutes at 37°C.
- Cool slides cool at room temperature for 5-7 minutes.
- Wash in room temperature PBS for 2 minutes (2X).
- Incubate slides in 0.2% Triton-X-100 solution in room
temperature PBS for 15 minutes.
- Wash slides in room temperature PBS for 2 minutes (2X).
Blocking:
- Incubate tissue slides in 3:1 methanol containing 3%
hydrogen peroxide for 30 minutes at room temperature.
- Wash with room temperature PBS for 2 minutes (2X).
- Incubate tissue in 10% animal serum for 1-2 hours
(depending on antibody). Serum must not be from same animal
as that from which antibody was prepared.
- Wash tissue slides with room temperature PBS for 2
minutes.
Primary Antibody Incubation:
- Incubate tissue with primary antibody at recommended
dilution either for 1 hour at room temperature or overnight
at 4°C.
- Wash with room temperature PBS for 5 minutes (2X).
Secondary Antibody Incubation/Developing:
- Prepare ABC reagents in the VectaStain Kit™ by first
adding 2 drops of reagent A and then 2 drops of reagent B to
5 mls PBS.
- Set solution at room temperature for 30 minutes.
- Prepare secondary antibody at 1:1000 dilution in
blocking serum which is prepared from the blue reagent in
VectaStain Kit™.
- Pipette solution containing the secondary antibody on to
the tissue sections and incubate at room temperature for 30
minutes.
- Wash slides with room temperature PBS for 2 minutes
(2X).
- Incubate tissue slides with ABC reagent (from Step 25)
for 30 minutes.
- Wash slides with room temperature PBS for 2 minutes
(2X).
- Prepare DAB working solution using the Peroxidase
Substrate DAB Kit™ (Vector Laboratories) by mixing 2.5 ml
distilled water, 1 drop of buffer solution, 2 drops of DAB,
and 1 drop of peroxidase.
- Stain tissues with DAB solution for approximately 1-2
minutes (or until brown stain develops, maximum 15 minutes).
- Rinse tissue slides in distilled H2O for 1 minute (1X)
and then 3 minutes (2X).
- Stain tissues with 1:3 dilution of Harris modified
Hematoxylin solution for 1-2 minutes.
- Rinse tissue slides in distilled H2O for 1
minute (1X), and 3 minutes (2X).
Dehydration & Mounting:
- Dehydrate tissue slides consecutively in 70%, 80%, and
90% ethanol for 2 minutes in each solution at room
temperature.
- Set slides in 100% ethanol for 3-5 minutes.
- Dunk tissue slides in Citrosolv™ for 2 minutes.
- Allow to dry for about 30 seconds, and mount with
VectaMount™ (Vector Laboratories).
Notes:
Protocol for Immunohistochemistry on Mouse Spine. Optimized
by The Van Den Berg Lab, by Azadeh Nasrazadani. Last Modified:
10/24/07