Successfully reported this slideshow.
DAKO IHC Staining Steps <ul><li>1.Deparaffinization and Rehydration </li></ul><ul><ul><li>2.Rinse with DW </li></ul></ul><...
Step1.Deparaffinization and Rehydration 1. Xylene - 5 mins. 2. Xylene - 5 mins. 3. Absolute ethanol -3 mins. 4. Absolute e...
Step 3.Target Retrieval & 4.Rinse Total: 15 – 18 mins. plus 20 mins. cooling time = 35-38 mins) 1. High - 5 mins. 2. Mediu...
5.Block Endogenous Peroxidase Hydrogen Peroxide 3% in TBST Hydrogen Peroxide blocking - 5 mins . (Wash buffer 1 min.)  Buf...
Primary Antibody Step 7.Primary Antibody 20 minutes
Step 8.Rinse with Buffer Buffer
Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 8  contd
Envision Step 9.Envision 15 minutes
Step 10.Rinse in Buffer Buffer
Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 10  contd
DAB 11.DAB 10 minutes
12.Rinse in DW DW d
Wash in DW 1 Minute DW
Hematoxylin 13.Hematoxylin 30 sec to 1 minute
14.Rinse in Tap Water
15.Coverslip with Aqeous medium
Interpretation of Results When >10% of total number of cancer cells stained by Ab = Positive
Trouble Shooting <ul><li>•  Possible causes for negative staining on positive slides: </li></ul><ul><li>1. Steps for the s...
Trouble shooting …… <ul><li>•  Possible causes for high background staining: </li></ul><ul><li>1. Endogenous peroxidase ac...
Trouble Shooting …… <ul><li>Possible causes for weak staining on all slides: </li></ul><ul><li>1. Specimen retains too muc...
 
Scoring ER/PR
Upcoming SlideShare
Loading in …5
×

Staining Steps3509

1,279 views

Published on

Customize these staining steps for your lab.

  • Be the first to comment

  • Be the first to like this

Staining Steps3509

  1. 1. DAKO IHC Staining Steps <ul><li>1.Deparaffinization and Rehydration </li></ul><ul><ul><li>2.Rinse with DW </li></ul></ul><ul><ul><li>3.Target Retrieval – Cool </li></ul></ul><ul><ul><li>4. Rinse in Tap Water and DW </li></ul></ul><ul><li>5.Block Endogenous Peroxidase </li></ul><ul><ul><li>6.Rinse with Buffer </li></ul></ul><ul><li>7.Primary Antibody </li></ul><ul><ul><li>8.Rinse with Buffer </li></ul></ul><ul><li>9.Envision </li></ul><ul><ul><li>10.Rinse with Buffer </li></ul></ul><ul><li>11.DAB </li></ul><ul><ul><li>12.Rinse with DW </li></ul></ul><ul><li>13.Hematoxylin </li></ul><ul><ul><li>14.Rinse with TW </li></ul></ul><ul><li>15.Coverslip with Aqueous Resin </li></ul>
  2. 2. 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.)
  3. 3. 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
  4. 4. 5.Block Endogenous Peroxidase Hydrogen Peroxide 3% in TBST Hydrogen Peroxide blocking - 5 mins . (Wash buffer 1 min.) Buffer Step 6 Rinse
  5. 5. Primary Antibody Step 7.Primary Antibody 20 minutes
  6. 6. Step 8.Rinse with Buffer Buffer
  7. 7. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 8 contd
  8. 8. Envision Step 9.Envision 15 minutes
  9. 9. Step 10.Rinse in Buffer Buffer
  10. 10. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 10 contd
  11. 11. DAB 11.DAB 10 minutes
  12. 12. 12.Rinse in DW DW d
  13. 13. Wash in DW 1 Minute DW
  14. 14. Hematoxylin 13.Hematoxylin 30 sec to 1 minute
  15. 15. 14.Rinse in Tap Water
  16. 16. 15.Coverslip with Aqeous medium
  17. 17. Interpretation of Results When >10% of total number of cancer cells stained by Ab = Positive
  18. 18. Trouble Shooting <ul><li>• Possible causes for negative staining on positive slides: </li></ul><ul><li>1. Steps for the staining procedure were not performed in correct </li></ul><ul><li>sequence. </li></ul><ul><li>2. Either primary or secondary antibody incubation steps were </li></ul><ul><li>skipped. </li></ul><ul><li>3. Destruction of labile antigens. </li></ul><ul><li>4. Improper fixation and/or processing of specimen. </li></ul><ul><li>5. Improper antigen recovery. </li></ul>
  19. 19. Trouble shooting …… <ul><li>• Possible causes for high background staining: </li></ul><ul><li>1. Endogenous peroxidase activity was not completely blocked. </li></ul><ul><li>2. Non-specific binding of protein to the specimen. Use protein block before primaryantibody. </li></ul><ul><li>3. Deparaffinization was not complete. </li></ul><ul><li>4. Excessive application of tissue adhesive. </li></ul><ul><li>5. Inadequate rinsing of slides. </li></ul><ul><li>6. Drying out of specimen during staining. </li></ul><ul><li>7. Over development of substrate. </li></ul>
  20. 20. Trouble Shooting …… <ul><li>Possible causes for weak staining on all slides: </li></ul><ul><li>1. Specimen retains too much liquid after rinsing steps. </li></ul><ul><li>2. Improper substrate preparation or use of old substrate </li></ul><ul><li>solution. </li></ul><ul><li>3. Deparaffinization was not complete (possibly accompanied by </li></ul><ul><li>high background). </li></ul>
  21. 22. Scoring ER/PR

×