Protocol for Immunolocalization on Sectioned Tissue
(K. Strissel)
1) Choose appropriate set of slides with sections. Allow slides to dry at room
temperature for 20-30 min (enough for multiple experimentals and good controls)
2) Draw a ring or box around sections on the slide with Pap-pen or use “probe clips” if
preferred – place slides in incubation box that contains moist paper (this aids in
preventing slides from drying out and in keeping small working volumes and
sections covered with adequate liquid).
3) Rehydrate sections by covering w/ 1X PBS for 5 min. Carefully add drops of PBS to
sections inside box or ring drawn on slide until sections are completely covered and
the solutions fills the ring.
4) Some proteins will require a permeabilization step. Typically, Triton X-100 is used
at 0.1% or 0.05% in 1X PBS. Incubate sections from 5-15min. Rinse 2-3 times with
1X PBS.
5) Carefully pour off the PBS and add the blocking solution. If using Vector ABC kit:
follow instructions provided there – this usually involves adding normal serum (3
drops/10mil) to 1X PBS and mixing. However, a more concentrated solution can be
used if the antibody is known to non-specifically bind to tissue: 3 drops normal
serum in 6 ml 1X PBS. Incubate at least 1 hour at room temperature or overnight at
4°C. Blocking solution should be kept on ice until needed.
6) Carefully pour off blocking solution, add PBS to rinse off once – add the primary
antibody (1°Ab) or control solution. Make sure sections are well-covered, incubate
at least 1 hour (rm. temp) or overnight (4°C).
7) Make up secondary antibody (2° Ab) as instructed in the kit: 10ml 1X PBS, 3 drops
normal serum, and 1 drop biotinylated 2° Ab. Vortex solution and keep on ice until
needed.
8) Remove 1° Ab and immediately add 1X PBS to rinse. Pour off the first PBS wash
and transfer slides to a Copeland Jar containing 1X PBS for the second wash. More
than one Copeland Jar can be used for the wash step. Additional washing can reduce
background staining. After each incubation step, the jar(s) should be changed with
fresh wash solutions. Remove slides and place back into incubation box – cover
slides immediately with 1X PBS. Pour off the last rinse of PBS just before adding
the secondary antibody.
Note: add and remove slides from the Copeland Jars slowly to prevent sections from
coming off the slides. This is more a problem with frozen sections.
9) Add the 2° Ab to the sections making sure to cover them completely. Incubate for
20-30 min.
10)
Mix up the ABC solution as instructed by the kit. The solution should be
mixed and allowed to sit for 30 minutes prior to use. This allows the Avitin-Biotin
complex to form. ABC solution: 5 ml 1X PBS buffer, +2 drops reagent A, mix. +2
drops reagent B, mix and keep on ice until needed.
11)
Wash off the 2° Ab in the same manner as the 1° Ab solution. Add the ABC
solution and incubate for 30 min.
12)
Wash off the ABC solution as before. After the last PBS wash, incubate the
sections in 10mM Tris ~pH 7.4. Keep sections covered with Tris buffer until the
substrate reaction solution is ready to be added.
13)
Mix up fresh substrate reaction solution: to 5ml of 10mM Tris buffer add 5
mg Diaminobenzadine(DAB) (a rough estimate is acceptable.) (gloves must be worn
and red bags must be used for disposal.) Mix, then remove any particulates from the
solution by filtration. Draw up the DAB solution into a 5ml syringe and push
through a 0.4 or 0.2 um filter into a fresh tube. Add ~40-50 ul or 1 drop of 3% H2O2.
For enhanced DAB staining solution, add 25 ul of NiCl2 solution (80mg/ml) to the
substrate reaction solution.
14)
Pour buffer off the slides and add the substrate reaction solution. The
substrate is usually deposited quickly so it is important to watch the sections, either
by eye or by microscope, and stop the reaction as soon as it is strong and clear. The
DAB solution will stain the substrate brown and the enhanced DAB solution will
give a purplish-black color. Avoid a non-specific reaction product on entire section.
When the reaction is done, place the slides into cold tap water in a Copeland Jar.
For: Immunofluorescent Localization:
After incubation in the fluorescent dye - conjugated 2° Ab, rinse 3 or 4 times with
PBS, drain off buffer, cover sections with 1 or 2 drops per slide of gel mount aqueous
mounting media. Place cover slips – allow media to “set” ~20 min – view slides.