LSBio

Immunohistochemistry Protocol for Formalin-Fixed, Paraffin Embedded Tissues Using Steam Based Antigen Retrieval

This protocol describes chromogenic immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue sections using heat-induced (steam) antigen retrieval in citrate buffer, with either an AP-Vector Red or an HRP-DAB detection system.

Tissue Sectioning, Deparaffinization and Rehydration

  1. Section paraffin blocks into 4 micron sections with a microtome and place on charged microscope slides (Fisher, ProbeOn, Cat. #22230900).
  2. Heat slides in a tissue-drying oven for 45 minutes at 60°C.
  3. Wash slides in 3 changes of xylene for 5 minutes each at room temperature.
  4. Wash slides in 3 changes of 100% alcohol for 3 minutes each at room temperature.
  5. Wash slides in 2 changes of 95% alcohol for 3 minutes each at room temperature.
  6. Wash slides in 1 change of 80% alcohol for 3 minutes at room temperature.
  7. Rinse slides in running distilled water for 5 minutes at room temperature.

Antigen Retrieval

  1. Steam slides in 0.01 M sodium citrate buffer, pH 6.0, for 20 minutes at 99–100°C.
  2. Remove slides from heat and hold in citrate buffer for 20 minutes at room temperature.
  3. Rinse slides in 1X TBS with Tween (TBST) for 1 minute at room temperature.

The following steps are conducted at room temperature. Do not allow tissues to dry at any time during the staining procedure.

Immunostaining with AP-Vector Red Detection System

  1. Apply Universal Protein Block (Dako, Cat. #X0909) to the slides and incubate for 20 minutes.
  2. Drain protein block from the slides.
  3. Apply diluted primary antibody to the slides and incubate for 45 minutes.
  4. Rinse slides in 1X TBST for 1 minute.
  5. Apply a biotinylated secondary antibody (specific to the host of the primary antibody) to the slides and incubate for 30 minutes.
  6. Rinse slides in 1X TBST for 1 minute.
  7. Apply Alkaline Phosphatase Streptavidin (Vector, Cat. #AK-5000) to the slides and incubate for 30 minutes.
  8. Rinse slides in 1X TBST for 1 minute.
  9. Apply Vector Red Alkaline Phosphatase Substrate (Vector, Cat. #SK-5100) to the slides and incubate for 30 minutes.
  10. Wash slides in distilled water for 1 minute.

Immunostaining with HRP-DAB Detection System

  1. Apply peroxidase block (3% hydrogen peroxide) to the slides and incubate for 5 minutes.
  2. Rinse slides in 1X TBST for 1 minute.
  3. Apply Universal Protein Block (Dako, Cat. #X0909) to the slides and incubate for 5 minutes.
  4. Drain protein block from the slides.
  5. Apply primary antibody to the slides and incubate for 45 minutes.
  6. Rinse slides in 1X TBST for 1 minute.
  7. Apply LSAB2 System-HRP Link solution (Dako, Cat. #K0679) to the slides and incubate for 15 minutes.
  8. Rinse slides in 1X TBST for 1 minute.
  9. Apply LSAB2 System-HRP Streptavidin-HRP solution (Dako, Cat. #K0679) to the slides and incubate for 10 minutes.
  10. Rinse slides in 1X TBST for 1 minute.
  11. Apply prepared DAB Substrate-Chromogen solution (Dako, Cat. #K3468) to the slides and incubate for 5 minutes.
  12. Rinse slides in 1X TBST for 1 minute.

Counterstaining with Hematoxylin

Stain slides with 65% Harris’ hematoxylin for 1 minute. Hematoxylin stains nucleic acids (nuclei) a deep blue-purple.

Dehydration and Coverslipping

Use this method only if the chromogen substrate is alcohol-insoluble (e.g. Vector Red or DAB).

  1. Wash slides in 2 changes of 80% alcohol for 1 minute each.
  2. Wash slides in 2 changes of 95% alcohol for 1 minute each.
  3. Wash slides in 3 changes of 100% alcohol for 1 minute each.
  4. Wash slides in 3 changes of xylene for 1 minute each.
  5. Apply a coverslip with a drop of permanent mounting medium.

For technical questions about LSBio protocols, contact technical@vectorlabs.com.