1. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
2. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes
Quench Endogenous Peroxidase:
1. Place slides in peroxidase quenching solution: 15-30 minutes. Add 3ml of 30% Hydrogen Peroxide to 200ml of Methanol.
2. Place slides in distilled water: 2 changes for 2 minutes each.
1. Preheat citrate buffer. Place 200ml of citrate buffer working solution into container, cover and place into steamer. Heat to 90-96C.
2. Place rack of slides into hot citrate buffer for 20 minutes. Cover.
3. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
4. Slowly add distilled water to further cool for 5 minutes.
5. Rinse slides with distilled water. 2 changes for 2 minutes each.
1. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen).
2. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
3. Drain the wash solution and apply 4 drops of blocking reagent to each slide and incubate for 15 minutes.
4. Drain blocking reagent (do not wash off the Blocking Reagent), apply 200ul of primary antibody solution to each slide, and incubate for 1 hour.
5. Wash slides with wash solution: 3 changes for 5 minutes each.
6. Drain wash solution, apply 4 drops of secondary antibody to each slide and incubate for 1 hour.
7. Wash slides with wash solution: 3 changes for 5 minutes each.
8. Drain wash solution, apply 4 drops of DAB substrate to each slide and develop for 5-10 minutes.
9. Wash slides with wash solution: 3 changes for 5 minutes each.
10. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes.
11. Wash slides with wash solution: 2-3 changes for 2 minutes each.
12. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
13. Rinse slides in distilled water.
14. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
15. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
16. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
17. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
18. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
19. Lay slides on a flat surface to dry prior to viewing under microscope.
-Use treated slides (e.g. HistoBond) to ensure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60C oven.
-All steps in which Xylene is used should be performed in a fume hood.
-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.
-200ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200ul may be used.
-5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1 minute for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired, increase the time (up to 10 minutes).