• All
  • Primary Antibodies
  • Conjugated Antibodies for IF
  • Conjugated Antibodies for FC
  • Secondary Antibodies
  • Antibody Labeling KitsNew
  • ELISA Kits
  • IHC Kits
  • Magnetic Cell Separation Kits
  • Cytokines & Growth Factors
  • Neutralizing/activating Antibodies
  • Nanobody-based Reagents
  • Accessory Products and Kits
  • Fusion Proteins
×
×
Host
0
Species Reactivity
0
Species Reactivity
0
Conjugate
0
Conjugate
0
Compatible Species
0
Conjugate
0

IHC On Paraffin Sections

Step-by-step protocol and tips for immunohistochemistry on paraffin-embedded sections.

Jump to:
Download PDF version

Preparing Fixed Tissues for Paraffin-Embedded Sectioning

Here we describe the procedure for preparing formalin-fixed paraffin-embedded tissue (FFPE) samples.

Fixation

For animal tissues, it is highly recommended to perform fixed perfusion before dissection to remove blood from the tissues to eliminate potential non-specific signals caused by endogenous IgGs. Flush the tissue with ice-cold PBS to remove blood, followed by perfusion with 4% PFA until adequate fixation is achieved. Alternatively, dissect the desired tissues (<3mm thick), wash with ice cold PBS, place into a cassette, and fix in 4% PFA or 10% formalin overnight at 4°C (but no longer than 24 hr). The adequate volume of fixative for fixation by immersion is typically 50-100x of the sample size. After fixation, wash tissues with running tap water for 30 minutes.

Dehydration

  • Move cassettes into 50% ethanol for 45 minutes.
  • Move cassettes into 70% ethanol for 45 minutes (Tissues can be left in 70% ethanol for long term storage, if required).
  • Move cassettes into 80% ethanol for 45 minutes.
  • Move cassettes into 95% ethanol for 45 minutes. Repeat this step a second time, using fresh 95% ethanol.
  • Move cassettes into 100% ethanol for 45 minutes. Repeat this step 2 more times, using fresh 100% ethanol each time.

Clearing

  • Move cassettes into Ethanol: Xylene (1:1 v/v) for 30 minutes.
  • Move cassettes into 100% Xylene for 30 minutes. Repeat this step a second time, using fresh 100% Xylene.

Wax Infiltration

  • Prepare beakers containing molten paraffin at 56-58°C.
  • Immerse cassettes into paraffin wax for 60 minutes. Repeat this step a second time, using fresh paraffin.

Embedding

  • Move cassettes onto an embedding station.
  • Using a mold made of thick plastic or stainless steel, coat the bottom layer of the mold with molten paraffin.
  • Place the tissues on top of the base wax, orienting them as desired. Partially cover with more paraffin. Place the cassette on top of the mold and completely fill the mold.
  • Place mold with tissues on a cold plate to solidify. The paraffin will solidify in 10-15 minutes.
  • Remove embedded tissues from the mold. Store at room temperature.

Sectioning Tissues on a Microtome

  • Section paraffin blocks at the desired thickness (usually 4-5 µm) on a microtome and transfer them to a 40°C water bath containing distilled water.
  • Transfer the sections onto charged slides coated with gelatin or poly-L-lysine. Place the slides in an oven to dry at 50-55°C for 4 hours (or 37°C for 6-8 hours).
  • Store slides at room temperature until ready for use.

Deparaffinizing and Rehydration (for FFPE sections only)

  • Immerse slides in xylene for 10 minutes. Repeat this step in fresh xylene for 10 minutes.
  • Rehydrate sections by sequentially incubating with 100%, 95%, 80%, and 60% ethanol for 5 minutes each.
  • Rinse sections with distilled water three times for 1-3 minutes each.

Antigen Retrieval (Optional)

  • Transfer slides to a microwave-proof container or a beaker on an electric stove and cover them with the recommended antigen retrieval buffer. If no antigen retrieval buffer is suggested, try TRIS-EDTA (pH 9) first. See “IHC Antigen Retrieval” section for more information.
  • Heat in the microwave for 10 minutes on medium power.
  • Allow slides to cool in the antigen retrieval buffer for approximately 35 minutes.

Quenching and Blocking

  • Rinse slides three times with 1x TBS for 3 minutes each.
  • Incubate slides with 3% H2O2 (diluted in distilled water) for 10 minutes to quench endogenous peroxidase activity. For FFPE samples, this step is often not necessary. See the section on “Quenching” for more information.
  • Rinse slides three times with 1x TBS for 3 minutes each.
  • Prepare 1% BSA or 5% normal blocking serum in 1x TBS. The serum should be derived from the same species in which the secondary antibody was raised. Block the sections for 30-60 minutes.