实验概要

The  following is a general procedure guide for preparation and staining of  acetone-fixed frozen tissues using a purified, unconjugated primary  antibody, biotinylated secondary antibody and streptavidin-horseradish  peroxidase (Sav-HRP) and DAB detection system. Because each antigen  differs in terms of requirement for fixation, amplification step, etc.,  it is not possible to write an inclusive protocol that will work for all  antigens. The user must determine optimal conditions for each antigen  of interest. Many protocols for staining individual antigens, as well as  useful tips and troubleshooting guides for immunohistochemistry, can be  found at the IHC World web site (http://www.ihcworld.com/).

实验步骤

Prepare frozen tissue sections (steps 1-8):

1.   Place a freshly dissected tissue block (<5 mm thick) on to a pre-labeled tissue base mold.

2.   Cover the entire tissue block with cryo-embedding media (e.g. OCT).

3.    Slowly place the base mold containing the tissue block into liquid  nitrogen till the en- tire tissue block is submerged into liquid  nitrogen to ensure tissue is frozen completely.

4.   Store the frozen tissue block at -80°C until ready for sectioning.

5.    Transfer the frozen tissue block to a cryotome cryostat (e.g. -20°C)  prior to sectioning and allow the temperature of the frozen tissue block  to equilibrate to the temperature of the cryotome cryostat.

6.   Section the frozen tissue block into a desired thickness (typically 5-10 µm) using the cryotome.

7.   Place the tissue sections onto glass slides suitable for immunohistochemistry (e.g. Superfrost).

8.   Dry the tissue sections overnight at room temperature. Sections can be stored in a sealed slide box at -80°C for later use.

Immunostain frozen tissue sections (steps 9-28):

9.    Fix the tissue sections with a suitable fixative.  One of the commonly  used fixation methods for frozen tissue sections is to immerse the  slides in pre-cooled acetone (-20°C) for 10 min.

10.  Pour off the fixative and allow acetone to evaporate from the tissue sections for > 20 min at room temperature.

11.  Rinse the slides in 300 ml of 10 mM phosphate buffered saline (PBS) at a neutral pH for 2 changes, 5 min each.

12.  Incubate the slides in 0.3% H2O2 solution in PBS at room temperature for 10 min to block endogenous peroxidase activity.

13.  Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

14.   (optional) Add 100 µl blocking buffer (e.g. 10% fetal bovine  serum  in  PBS) onto the sections of the slides and incubate in a humidified  chamber at room temperature for 1 h.

15.  Drain off the blocking buffer from the slides.

16.   Apply 100 µl an appropriately diluted primary antibody (in antibody  dilution buffer, e.g. 0.5% bovine serum albumin  in PBS) to the sections  on the slides and incubate in a humidified chamber for 1 h at room  temperature or overnight at 4°C.

17.  Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

18.   Apply 100 µl an appropriately diluted biotinylated secondary antibody  (using the anti- body dilution buffer) to the sections on the slides and  incubate in a humidified chamber at room temperature for 30 min.

19.  Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

20.   Add 100 µl pre-diluted Sav-HRP conjugates (using the antibody dilution  buffer) to the sections on the slides and incubate in a humidified  chamber at room temperature for 30 min (keep protected from light).

21.  Rinse the slides in 300 ml PBS for 2 changes, 5 min each.

22.  Apply 100 µl DAB substrate solution (freshly made just before use: 0.05% DAB - 0.015% H₂O₂  in PBS) to the sections on the slides to reveal the color of the  antibody staining. Allow the color development for < 5 min until the  desired color intensity is reached. (Caution: DAB is a suspected  carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)

23.  Wash slides in 300 ml PBS for 2 changes 5 min each.

24.  (optional) Counterstain slides by immersing sides in Hematoxylin (e.g. Gill’s Hema- toxylin) for 1-2 min.

25.  Rinse the slides in running tap water for > 15 min.

26.  Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100% and 100%), 5 min each.

27.   Clear the tissue slides in 3 changes of xylene and coverslip using  mounting solution (e.g. Permount). The mounted slides can be stored at  room temperature permanently.

28.  Observe the color of the antibody staining in the tissue sections under  microscopy.