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DAKO IHC Staining Steps 1.Deparaffinization and Rehydration

2.Rinse with DW

3.Target Retrieval – Cool 4. Rinse in Tap Water and DW

5.Block Endogenous Peroxidase 6.Rinse with Buffer

7.Primary Antibody 8.Rinse with Buffer

9.Envision 10.Rinse with Buffer

11.DAB 12.Rinse with DW

13.Hematoxylin 14.Rinse with TW

15.Coverslip with Aqueous Resin

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Step1.Deparaffinization and Rehydration

1. Xylene - 5 mins.2. Xylene - 5 mins.

3. Absolute ethanol -3 mins.4. Absolute ethanol - 3 mins

5. 95% ethanol - 3 mins.6. 95% ethanol - 3 mins.

Step 2 Rinse Distilled water (1 – 5 min.)

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Step 3.Target Retrieval & 4.Rinse

Total: 15 – 18 mins. plus 20 mins. cooling time = 35-38 mins)1. High - 5 mins.2. Medium High/Low - 5 mins.3. Medium - 5 mins.4. Medium - 3 mins.5. Cooling time - 20 mins.

700-800 watts microwave oven

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5.Block Endogenous Peroxidase

Hydrogen Peroxide 3% in TBST

Hydrogen Peroxide blocking - 5 mins.

(Wash buffer 1 min.)

Buffer

Step 6 Rinse

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Primary Antibody

Step 7.Primary Antibody

20 minutes

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Step 8.Rinse with Buffer

Buffer

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Buffer BufferBuffer

Wash in Buffer x 3

Dip 10 times Dip 10 times 1 minute

Step 8 contd

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Envision

Step 9.Envision

15 minutes

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Step 10.Rinse in Buffer

Buffer

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Buffer BufferBuffer

Wash in Buffer x 3

Dip 10 times Dip 10 times 1 minute

Step 10 contd

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DAB

11.DAB

10 minutes

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12.Rinse in DW

DW

d

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Wash in DW

1 Minute

DW

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Hematoxylin

13.Hematoxylin

30 sec to 1 minute

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14.Rinse in Tap Water

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15.Coverslip with Aqeous medium

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Interpretation of Results

When >10% of total number of cancer cells stained by Ab = Positive

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Trouble Shooting• Possible causes for negative staining on

positive slides:• 1. Steps for the staining procedure were not

performed in correct• sequence.• 2. Either primary or secondary antibody

incubation steps were• skipped.• 3. Destruction of labile antigens.• 4. Improper fixation and/or processing of

specimen.• 5. Improper antigen recovery.

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Trouble shooting ……

• • Possible causes for high background staining:

• 1. Endogenous peroxidase activity was not completely blocked.

• 2. Non-specific binding of protein to the specimen. Use protein block before primaryantibody.

• 3. Deparaffinization was not complete.• 4. Excessive application of tissue adhesive.• 5. Inadequate rinsing of slides.• 6. Drying out of specimen during staining.• 7. Over development of substrate.

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Trouble Shooting ……

• Possible causes for weak staining on all slides:

• 1. Specimen retains too much liquid after rinsing steps.

• 2. Improper substrate preparation or use of old substrate

• solution.• 3. Deparaffinization was not complete

(possibly accompanied by• high background).

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Scoring ER/PR