Start Right to Finish Well—The Importance of the Sample Preparation Process in Your IHC and IF Workflow

Popular wisdom says that finishing well is more important than how you start. But when it comes to immunohistochemistry (IHC) and immunofluorescence (IF), starting well is essential to great results. It’s crucial to thoroughly consider and plan the sample preparation process if you want clear and crisp immunostaining at the end.

Vector Laboratories created an in-depth Staining Sample Preparation Guide that provides an overview of the sample preparation process and offers guidance on making decisions and troubleshooting for each step. The different stages in the sample preparation process—storage, sectioning, deparaffinization and rehydration, antigen retrieval, and hydrophobic barrier application (Figure 1) — vary according to the tissue preservation method, which in turn depends on the final application. Thus, reading through the entire guide before starting your next IHC/IF experiment is a wise way to start strong and ensure excellent results.

Here’s a sneak peek of what you’ll find in the Staining Sample Preparation Guide.

Sample Storage

After collecting the specimen, you have 2 choices for long-term tissue preservation—paraffin-embedding and cryopreservation. The formalin-fixation, paraffin-embedding (FFPE) process has several steps with specific purposes:

  • Formalin fixation helps to inhibit biochemical reactions in the tissue, prevents autolysis and putrefaction, and forms covalent bonds to help preserve tissue morphology.
  • Dehydration uses a series of graded alcohols (from a lower to a higher concentration) to replace the fixative with alcohol.
  • Clearing with xylene (or another organic solvent) clears the alcohol.
  • Paraffinization impregnates the tissue with warm paraffin that hardens to create an FFPE block as it cools down.

Cryopreservation is a simpler and quicker process than FFPE, and you can choose from 2 options when working with this approach:

  • Snap-freeze the fresh tissue in liquid nitrogen, isopentane, or dry ice; mount it in a block of OCT (optimal cutting temperature) compound; section it on a cryostat; and fixate it with one of the many choices of fixatives (e.g., acetone, methanol, ethanol, formaldehyde).
  • Fixate the tissue; impregnate it with a cryoprotectant solution; and freeze it in liquid nitrogen, isopentane, or dry ice.
Figure 1. Sample preparation process. A. Options for tissue preservation include formalin-fixed, paraffin-embedded (FFPE) or frozen block. B. Tissue blocks can be sectioned in different thicknesses using different equipment according to the final application. C. A series of washes in xylene ensures complete paraffin removal. Washes in graded alcohol gradually replace xylene for water*. D. Heat-induced epitope re¬trieval promotes breakage of cross-links formed during fixation and unmasking of antigen**. E. PAP pen applies a hydrophobic barrier around the tissue section, containing reagents to a small area. *Deparaffinization and rehydration steps are only applicable when working with FFPE tissue sections. **Heat-induced epitope retrieval is necessary when working with tissue that was snap-frozen right after dissection. Anti¬gen retrieval improves staining in specimens that underwent formalin fixation regardless of the subsequent choice of storage being paraffinization or cryopreservation.

What’s the right option for your experiments? It depends on the antigen in question, the final application, and some other factors. For example, FFPE is ideal for experiments that require very thin sections for higher microscopic resolution. Frozen blocks retain better antigenicity and are suitable for preserving highly sensitive proteins, post-translational modifications, enzymatic activity, and nucleic acids.

The Staining Sample Preparation Guide provides a detailed comparison of the pros and cons of the 3 available options for tissue preservation. It also highlights the steps that allow long-term storage and provides specific recommendations (temperature and length of storage) for ideal tissue preservation. The guide also includes a list of applications that can benefit from fixatives other than formalin (e.g., acetone, methanol, ethanol). One final bonus tip you will find under the sample storage section of the guide are 2 methods for quickly freezing specimens to avoid the formation of large ice crystals.


After choosing the best approach to preserve your specimen, you need to tackle tissue sectioning. The main decision required for this step is how thick you should cut your sections, and the 3 specific needs that can influence your choice are:

  • Microscopic resolution—as mentioned in the previous section, thinner sections yield a higher resolution
  • Morphological assessment—experiments that require analysis of 3D structures benefit from thicker sections
  • Stereological quantification—quantifying the total number of cells in a particular region requires even thicker sections

After getting your tissue slices on the slide, you typically need to allow them to air-dry for about 24 hours. However, the sample preparation guide provides some tips & tricks on how to shorten this step for a quicker drying process. It is critical to properly dry the sections to prevent detachment downstream in the workflow. If you constantly face this problem, you might benefit from using chemical adhesive reagents, such as VECTABOND® Reagent, Tissue Section Adhesion.

Deparaffinization and Rehydration

If you are working with FFPE sections, you need to remove the paraffin and rehydrate the tissue, or all the aqueous solutions downstream in the process won’t penetrate the specimen. This step is the exact opposite of the dehydration and paraffin impregnation process:

  • Xylene removes paraffin
  • A series of graded dilutions of alcohol—100%, 95%, 70%—removes xylene and rehydrates the tissue
  • A final wash in water ensures that no alcohol is left in the tissue

It’s crucial to use fresh reagents and allow adequate incubation time in each solution, or you might get uneven or patchy staining at the end. Also, note that thicker sections require longer incubation and wash times.

Antigen Retrieval

If you opt to use a formalin-based fixative, you likely need to include the antigen retrieval step in the IHC/IF workflow. During fixation, formalin promotes the formation of cross-links between amino acids of proteins in the specimen, which helps to harden the tissue and preserve overall morphology. But those cross-links also prevent the primary antibody from binding to the target epitope. You can use a proteolytic approach or heat-induced epitope retrieval (HIER) to break down the cross-links and unmask the antigen, but the latter yields better results (Figure 2).

The HIER process comprises 4 variables that you need to empirically determine—heat source, buffer pH, temperature, and incubation time. When performing antigen retrieval, start by checking the antibody manufacturer’s recommendation for the ideal retrieval buffer and temperature. Also, check the Staining Sample Preparation Guide as it discusses the many options available and how to make changes when necessary.

Hydrophobic Barrier Pen

Congratulations! You’ve reached the end of the sample preparation process, and all that is left to do is to apply a hydrophobic barrier around the sections. Creating a barrier around the section allows for the use of different reagents on the same slide as well as a need for a smaller volume of solution to cover the sections, this in turn has the added benefits of flexibility and cost reduction for your experiments. In the past, nail polish was the go-to approach for completing this step, but using a ImmEdge® Hydrophobic Barrier PAP Pen is a much easier way of creating a hydrophobic barrier.

Remember, in the IHC/IF world, starting well is essential to great results. If you want to learn more about sample preparation and gather additional information to support your decision-making, download a copy of the Staining Sample Preparation Guide. If you need further support, reach out to our Technical Support team. We’re here to help you start and finish strong!

Figure 2. Heat-induced epitope retrieval process. During fixation, formalin-based fixatives promote the formation of methylene cross-links between protein residues. This process promotes tissue hardening and preservation but also masks target antigens and prevents antibody binding. Heating the specimen in a defined buffer breaks the cross-link and exposes the epitope.