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Preparation and Chromogenic IHC Staining of Frozen Tissue Sections

Creative Biolabs has developed and optimized the following protocol for chromogenic immunohistochemistry (IHC) staining experiments using frozen tissue sections. This protocol provides a basic guide for the fixation, cryostat sectioning, and staining of frozen tissue samples. Please read Protocol of Immunohistochemistry (IHC) in its entirety before beginning.

Contents

Step 1-1: Tissue Preparation-Perfusion and Fixation

Step 1-2: Tissue Preparation-Cryopreservation

Step 2: Blocking Non-specific Binding

Step 3: Antibody Staining

Step 4: Detection

Step 5: Dehydration and Mounting

Reagents

Methods

Note: Step 1 can be skipped if you are working with pre-mounted slides.

Step 1-1: Tissue Preparation-Perfusion and Fixation

  1. Fix tissue by perfusing the animal with freshly prepared 4% paraformaldehyde or by immersing it in 4% paraformaldehyde for 4-24 hours at RT.

    Note: Fixation time and temperature need to be optimized depending on the tissue type and size.

  2. Cryoprotect the tissue by directly perfusing a sucrose solution.
  3. Embed tissue in OCT cryostat sectioning medium and store at -80°C until ready for sectioning.

    Note: Tissue can be safely stored for 6-12 months.

  4. When ready for sectioning, move the embedded tissue directly into the cryostat and use OCT medium to mount it to the chuck. Allow the temperature of the tissue to equilibrate with the cryostat.
  5. Cut the tissue in 5-20 µm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides by placing the cold sections onto warm slides.

    Note: Slides can be safely stored for 6-12 months at -80°C until ready for staining.

Step 1-2: Tissue Preparation-Cryopreservation

  1. After dissection, immediately snap freeze tissue with isopentane cooled by liquid nitrogen. After the tissue is frozen, place it in dry ice and move to -80°C until ready for cutting.
  2. Embed tissue in OCT compound by slowly layering the compound so that the tissue does not thaw. Move the embedded tissue directly into the cryostat and use OCT medium to mount it to the chuck. Allow the temperature of the tissue to equilibrate with the cryostat.
  3. Cut the tissue in 5-20 µm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides by placing the cold sections onto warm slides.

    Note: Slides can be safely stored for 6-12 months at -80°C until ready for fixing. Uncut tissue can be restored at -80°C.

  4. Remove slides from freezer and fix with cold fixative for 10 minutes. Proceed to staining.

Step 2: Blocking Non-specific Binding

  1. Warm slides to RT. Wash slides twice with PBS.

    Note: Fixation may result in epitope masking and non-specific background that can impact specific labeling. If necessary, a protocol for antigen retrieval can be performed at this time.

  2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen.
  3. Wash the sections twice for 10 minutes with 1% animal serum in PBS-T.

    Note: The species of the animal serum used in blocking buffers is dependent on the host of your secondary antibody. For instance, when using a goat anti-mouse secondary, use goat serum.

  4. Block non-specific binding by incubating the tissue sections with 5% serum in PBS-T for 30 minutes at RT.

Step 3: Antibody Staining

  1. Add the primary antibody diluted in 1% animal serum in PBS-T to the sections. Incubate at RT for 1-2 hours. Continue the incubation overnight at 4°C.

    Note: The recommended starting dilution is 2-5 µg/ml if there are not specified antibody dilution.

  2. Wash sections twice with 1% serum PBS-T for 10 minutes each.
  3. Add a HRP conjugated (HRP-DAB detection method) or biotinylated secondary antibody (ABC-HRP-DAB detection method) to each section. Incubate the sections at RT for 1 hour.
  4. Wash sections twice with 1% serum PBS-T for 10 minutes each.

Step 4: Detection

  1. For the ABC method, add ABC-HRP reagent. Incubate at RT for 1 hour.
  2. Wash sections twice in PBS for 10 minutes each.
  3. Prepare a working solution of DAB and apply to tissue sections.

    Note: Monitor the reaction as the chromogenic reaction turns the epitope sites brown. Time of color development may vary from few seconds to 10 minutes.

  4. As soon as the section develop, immerse them in deionized water for 2 minutes each.
  5. If nuclear counterstaining is desired, use Hematoxylin according to the manufacturer’s instructions.

If you are using an aqueous chromogen instead of DAB such as Fast Red, AEC, skip the following Step 5 and mount in aqueous media instead of organic mounting media.

Step 5: Deparaffinization and Mounting

  1. Dehydrate tissue sections by moving slides through the following solutions twice for 2 minutes each: 95% Ethanol-100% Ethanol-Xylene.
  2. Add mounting media to slides and top with coverslips. The DAB reaction is permanent and stable and can be analyzed under a brightfield microscope at any time.

Related Sections

Creative Biolabs presents a presentation that will be invaluable to beginners in IHC and act as a fact-packed synopsis for those of you interested in teaching others about the virtues of this powerful application. To further assist learning, we have introduced the following sections for your viewing. Please refer to the corresponding section for details.