Protocol

Preparing Paraffin Tissue Sections for Immunostaining

This protocol was adapted from “Staining Tissues,” Chapter 6, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

INTRODUCTION

Most histological studies are carried out on paraformaldehyde-fixed, paraffin-embedded tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is immediately informative. The fixation and embedding procedures are harsh, however, and many antigens are not well preserved. Be aware that prolonged fixation diminishes the ability of antibodies to work, and in pathology laboratories, the duration and other details of fixation are rarely standardized. In addition, the constant rate of penetration of fixative into tissue means that not all parts of a sample will be fixed in the same way. To minimize variation in immunostaining properties, it is best to attend to specimen collection and fixation personally.

MATERIALS

Reagents

Ethanol (absolute and 95%)

Fixative, such as Paraformaldehyde in PBS (4%, prepared fresh), Bouin’s fixative, or Methocarn (see Step 2)

Avoid mercury-containing fixative absolutely.

Tissue to be immunostained

Xylene

Equipment

Microtome

Paraffin embedding equipment

METHOD

  • 1. Cut small blocks of tissue ~1 cm2 × 0.4 cm.

  • 2. Place the tissue blocks in fixative. Freshly prepared 4% paraformaldehyde in PBS is recommended for samples that will be used solely for immunochemical localization either directly or with counter- or double stains, but a broad range of fixatives (including Bouin’s and methocarn) can be used.

  • 3. Incubate the samples for 2 hours to overnight.

    Fixatives penetrate tissues at a rate of ~1 mm per hour. Use this number to estimate the minimum incubation time.

  • 4. Follow standard paraffin embedding procedures as suggested by the makers of the microtome. Paraffin blocks can be stored for years.

  • 5. Collect 4-μm sections onto clean glass slides.

  • 6. Incubate the sections overnight at 37ºC.

    Higher temperatures tend to cause loss of many epitopes and should be avoided if possible.

  • 7. Dewax the sections in xylene. Change two times, 3 minutes each.

  • 8. Rehydrate by passing the sections through graded alcohols (two changes of absolute ethanol followed by two changes of 95% ethanol, 3 minutes each).

    If methocarn was used to fix the tissues, the first step of rehydration must use anhydrous ethanol.

  • 9. Rinse in H2O.

  • 10. Samples are now ready for the application of antibodies (Binding Antibodies to Tissue Sections).

    Because of the harsh fixation, embedding, and preparation conditions, staining of paraffin-embedded tissue sections usually requires sensitive detection methods and may need amplification using multiple-layer techniques.

TROUBLESHOOTING

Problem: Endogenous peroxidase activity interferes with detection

Solution: To block endogenous peroxidase activity, incubate the specimen with a solution of 4 parts of methanol to 1 part of 3% H2O2 for 20 minutes. For specimens such as spleen or bone marrow containing high peroxidase activities, better results may be obtained by using a solution of 0.1% phenylhydrazine hydrochloride in PBS. Although some sources suggest the simple application of 3% H2O2 to the specimen, this should not be done because violent reactions and bubble formation can destroy the specimen. Blocking procedures may harm some antigens, so it may be easier to change the detection reagent than to block endogenous enzyme activities. (Note: Phenylhydrazine hydrochloride is highly toxic and is a carcinogen. Avoid inhalation, ingestion, or skin absorption.)

Problem: Endogenous alkaline phosphatase activity interferes with detection

Solution: To block endogenous alkaline phosphatase activity, include 0.1 mM Levamisole in the substrate solution. This inhibitor does not diminish the activity of intestinal alkaline phosphatase used to prepare the labeled antibody but does inhibit the activity of other tissue phosphatases. Alkaline phosphatase-labeled antibodies should not be used to stain specimens containing endogenous intestinal alkaline phosphatase. Blocking procedures may harm some antigens, so it may be easier to change the detection reagent than to block endogenous enzyme activities.

A more recent Protocol discussing this method is available

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