Immunohistochemistry Protocol
After tissue samples are removed from a mouse, sections for the tissue samples are made
to be studied under a microscope. Immunohistochemistry utilizes different combinations
of antibodies to fluoresce target cells.
Solutions:
1X PBS
Dilute 10X stock to 1X
1X TBS
Mix 400 ml of ddH2O, 50 ml of 10X NaCl, and 50 ml of 10X (0.5M) Tris-Cl (pH 7.6)
0.3% Triton X-100 Solution
Dilute 1.2 ml of Triton X-100 solution into 400 ml of 1X PBS. Heat in microwave for 20
seconds and stir for twenty minutes before use.
Blocking Solution
Prepare 1.5%-3.0% goat or horse serum in 1X PBS. Sigma blocking serum found in
small -20 °C freezer.
0.3% Quenching Solution
Dilute 2 ml of 30% Hydrogen Peroxide in 200 ml MeOH.
ABC Solution
Add on drop of Solution A and one drop of Solution B for every 5 ml of 1X PBS. Vortex
after adding each solution. Make 30 minutes before use. Kit found in small 4 °C
refrigerator.
DAB Solution
Using Signet Kit: Use one drop of DAB chromagen solution for every 1 ml of DAB
substrate solution. Found in small 4 °C refrigerator.
Antigen Retrieval Solution
Dilute 10X AR-10 solution to 1X. Found in small 4 °C refrigerator.
Procedure – solution amounts are for approximately 20 slides
Day 1
1.
2.
3.
4.
Warm slides to 58 °C for 30 min.
Cool slides to room temperature for 10 min.
Make 2000 ml 1X PBS
Deparrafinize Sections
a)
b)
c)
d)
e)
f)
100% Xylene, 3 times for 3 min.
100% Ethanol, 3 times for 1 min.
95% Ethanol, 1 min
70% Ethanol, 1 min
50% Ethanol, 1 min
30% Ethanol, 1 min
*Make sure no air bubbles remain on slides when dipping in solution.
*When Xylene and EtOH jars seem overused, discard solution in jar (1) and rotate other
jars forward with jar (3) having fresh solution added to it.
*Pour diluted EtOH from bottle and return to bottle after use.
5. Wash slides in 1X PBS, 2 times for 5 min.
*May be stored in 1X PBS for long periods of time.
*Prepare Triton X-100 Solution
*Prepare Antigen Retrieval Solution
Antigen Retrieval Procedure
6. Take slides out of 1X PBS and wash in distilled water, 3 times for 1 min.
7. Fill up coplin jar with Antigen Retrieval Solution.
8. Place slides in coplin jar, with one jar containing only AR solution.
*Don’t allow tissue samples to face each other in coplin jar.
9. Heat coplin jars:
a) 2 min. @ default power level or until all jars have boiled.
b) 5 min. @ power level 2.
c) Check AR solution level in jars, refill AR solution in jars with slides from
slideless coplin jar.
d) Bring temperature of slides back to boiling by heating 10 to 15 seconds @ default
power level.
e) Repeat step (b).
10. Remove coplin jars from microwave and let sit for at least one hour at room
temperature.
*Slides must be allowed to cool gradually to prevent peeling and/or wrinkling of tissue.
11. Wash slides in distilled water 3 times for 1 min.
12. Wash slides in 1X PBS, 5 min.
13. Permeabilize slides in 0.3% Triton X-100, 2 times for 10 min.
*Approximately 200 ml of solution needed for each glass jar.
*Use this time to prepare blocking solution for blocking the sections as well as for the
primary antibody. Blocking serum depends on the primary antibody
14. Wash slides in 1X PBS, 3 times for 5 min.
15. Take slides out of 1X PBS and dry with kimwipe, be careful not to touch the tissue.
16. Encircle tissue sections with PAP pen.
17. Incubate tissue sections in blocking solution for 30 min. at room temperature
*Make sure that at any time during this procedure the slides do not dry, this will cause
wrinkling. This may be avoided by drying and blocking each slide individually in
humidified chamber. Slides may remain in blocking solution for up to an hour.
18. Aspirate Blocking Solution and apply primary antibody (diluted in Blocking
solution). Dilution of primary antibody is specific to the antibody.
19. Incubate section with primary antibody overnight in 4 °C.
Day 2
1. Save primary antibody by pouring off slides.
*Primary antibody can be saved up to one month when dilution of Sodium Azide (1:100)
is added.
2.
3.
4.
5.
Wash slides in 1X PBS, 3 times for 5 min.
Take slides out of wash and aspirate off 1X PBS.
Prepare Quenching solution.
Apply a 1:200 dilution of secondary antibody (in blocking solute) to the slides for one
hour at room temperature in the dark.
6. Repeat step 2.
7. Prepare ABC solution.
8. Quench out endogenous peroxidase activity in quenching solution at room
temperature for 30 min.
9. Repeat step 2.
10. Dry and aspirate slides.
11. Incubate slides in ABC solution at room temperature for 30 minutes.
12. Repeat step 2.
13. Make 10 ml DAB solution and place on ice and in foil.
14. Perform color reaction for control section:
a) Add 400 μl DAB solution to section.
b) Cover slide for 5 min.
c) Stop reaction by placing slide in 1X PBS.
d) Observe slide under microscope for background and staining of target cells.
e) Adjust time for color reaction relatively according to control staining (perform
color reaction again if necessary).
15. Perform color reaction for rest of slides using control time.
16. Wash slides in 1X PBS for 10 min.
17. Wash in distilled water for 5 min.
18. Counterstain slides:
a) 5 dips in hematoxylin
b) rinse in running tap water until clear again
c) clarifier solution for 1 min.
d) rinse in tap water for one min.
e) blueing solution for one min.
f) rinse in tap water for one min.
g) 10 dips in 95% EtOH
h) 100% EtOH 3 times for one min.
i) 100% Xylene 3 times for one min.
19. Place cover slip on slide using DPX solution
*Free slide of bubbles while applying cover slip by slowly slipping cover slip from
bottom of slide.