1. Cut and mount sections on slides coated with a suitable tissue adhesive.
2. Deparaffinise sections and rehydrate to distilled water.
3. Place sections in 0.5% hydrogen peroxide/methanol for 10 minutes (or use other appropriate endogenous peroxidase blocking
procedure). Wash sections in tap water.
4. Heat 1500ml of the recommended unmasking solution (0.01M citrate buffer, pH6.0 unless otherwise indicated overleaf) until
boiling in a stainless steel pressure cooker. Cover but do not lock lid.
5. Position slides into metal staining racks (do not place slides close together as uneven staining may occur) and lower into
pressure cooker ensuring slides are completely immersed in unmasking solution. Lock lid.
6. When the pressure cooker reaches operating temperature and pressure (after about 5 minutes) start a timer for 1 minute (unless
otherwise indicated on the data sheet).
7. When the timer rings, remove pressure cooker from heat source and run under cold water with lid on. DO NOT OPEN LID UNTIL
THE INDICATORS SHOW THAT PRESSURE HAS BEEN RELEASED. Open lid, remove slides and place immediately into a
bath of tap water.
8. Wash sections in TBS* buffer (pH 7.6) for 1 x 5 minutes.
9. Place sections in diluted normal serum (or RTU Normal Horse Serum) for 10 minutes.
10. Incubate sections with primary antibody.
11. Wash in TBS buffer for 2 x 5 minutes.
12. Incubate sections in an appropriate biotinylated secondary antibody.
13. Wash in TBS buffer for 2 x 5 minutes.
14. Incubate slides in ABC reagent (or RTU streptavidin/peroxidase complex).
15. Wash in TBS buffer for 2 x 5 minutes.
16. Incubate slides in DAB or other suitable peroxidase substrate.
17. Wash thoroughly in running tap water.
18. Counterstain with haematoxylin (if required), dehydrate and mount.
SOLUTIONS
1. 0.01M CITRATE BUFFER (pH 6.0)
Add 3.84 grams of citric acid (anhydrous) to 1.8 litres of distilled water. Adjust to pH 6.0 using concentrated NaOH. Make up to 2
litres with distilled water.
2. 1mM EDTA (pH 8.0)
Add 0.37g of EDTA (SIGMA product code E-5134) to 1 litre of distilled water. Adjust pH to 8.0 using 1.0M NaOH.
3. 20mM TRIS/0.65mM EDTA/0.0005% TWEEN (pH9.0)
Dissolve 14.4g Tris (BDH product code 271197K) and 1.44g EDTA (SIGMA product code E-5134) to 0.55 litres of distilled water.
Adjust pH to 9 with 1M HCI and add 0.3ml Tween 20 (SIGMA product code P-1379). Make up to 0.6 litres with distilled water. This
is a 10x concentrate which should be diluted with distilled water as required (eg 150ml diluted with 1350ml of distilled water).
* In most applications, 10mM phosphate, 0.15 M NaCl, pH 7.6 (PBS) can be used instead of 50 mM Tris, 0.15 M NaCl, pH 7.6
(TBS).
SAFETY NOTE
To ensure the correct and safe use of your pressure cooker, PLEASE READ MANUFACTURER’S INSTRUCTIONS.