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Retrievals

Immunohistochemistry

The Immunohistochemistry staff furnishes the protocols for heat-induced epitope retrieval and enzymatic epitope retrieval.

Heat-Induced Epitope Retrieval

Decloaker

  1. Add 500 ml of distilled water to the pan of the Decloaker™.
  2. Place a full rack of slides into a Tissue Tek® container with 200 ml of retrieval solution.
  3. Place the container stably inside the pan and decloak for 5 minutes at 120°C (or another time and temperature if deemed necessary). Insert blank slides into any empty slots in the rack to ensure even heating of slides.
  4. Depressurize for 10 minutes.
  5. Remove the pan top and cool for 10 minutes.
  6.  Rinse the slides in 2 changes of distilled water for 3 minutes each time.

 

Equipment information: Decloaker

 

Microwave (Panasonic Model No. NN-S744WF 1200 Watts)

  1. Place a full rack of slides into a Tissue Tek® container with 200 ml of retrieval solution
  2. Microwave the slides for 5 minutes at power level 5.
  3. Cool the slides for 1 minute, adding additional retrieval solution if necessary.
  4. Microwave the slides again for 5 minutes at power level 5.
  5. Remove the slides from the microwave and cool for 20 minutes at room temperature.
  6. Rinse the slides in 2 changes of distilled water for 3 minutes each time.

 

Equipment information: Microwave 

 

Steamer

  1. Add distilled water to the bottom portion of the steamer.
  2. Preheat 200 ml of retrieval solution in a Tissue Tek® container in the steamer between 95 C and 100 C.
  3. Immerse a full rack of slides into the retrieval solution and place the lid back on the steamer.  Insert blank slides into any empty slots in the rack to ensure even heating of slides.
  4. Steam the slides for 20 minutes.
  5. Remove the container from the steamer and cool for 20 minutes.
  6. Rinse the slides in 2 changes of distilled water for 3 minutes each time.

 

Equipment information: Steamer

 

Enzymatic Epitope Retrieval

Pepsin

  1. Note: The user should validate the most optimal enzyme concentration and incubation time for their stain.
  2. Preheat slides to 37ºC in 1X Wash Buffer.
  3. Incubate the slides in Carezyme II: Pepsin (predilute) for 3 minutes at 37ºC. (Allow the pepsin to reach room temperature before use.)
  4. Rinse the slides in distilled water for 1 minute to stop the enzymatic reaction.

 

Protease

  1. Note: The user should validate the most optimal enzyme concentration and incubation time for their stain.
  2. Preheat slides to 37ºC in 1X Wash Buffer.
  3. Prepare a 0.01% protease solution by dissolving the appropriate amount of powder in distilled water.
  4. Incubate the slides in the protease solution for 5 minutes at 37ºC.
  5. Rinse the slides in distilled water for 1 minute to stop the enzymatic reaction.

 

Proteinase K

  1. Note: The user should validate the most optimal enzyme concentration and incubation time for their stain. This protocol is specifically for the TUNEL stain.
  2. Prepare a 1:10 dilution (20 μg/ml) of proteinase K in 1X PBS buffer.
  3. Incubate the slides for 15 minutes at room temperature.
  4. Rinse the slides in distilled water for 2 minutes to stop the enzymatic reaction.

 

Trypsin

  1. Note: The user should validate the most optimal enzyme concentration and incubation time for their stain. We typically do 0.01% or 0.1%, depending on the stain.
  2. Prepare a trypsin solution by dissolving the appropriate amount of powder in 0.05M Tris-HCl • CaCl2 solution. Do not add the solution to the trypsin any earlier than 5 minutes prior to incubation. Trypsin looses 75% of its reactivity within 30 minutes at 37ºC.
  3. Incubate the slides in the trypsin solution for 20 minutes at 37ºC.
  4. Rinse the slides in distilled water for 1 minute to stop the enzymatic reaction.

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