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Sample Preparation For Immunohistochemistry

The preparation of samples for Immunohistochemistry (IHC) is a determining step in the process of these immunoassays; each tissue must be properly collected and prepared according to each study.

The preparation of these samples should be done according to the fixation method used, which in turn will depend on the detection technique chosen.

In this post we present you a brief guide with the keys to sample preparation for Immunohistochemistry.

The samples for immunohistochemistry can be prepared as sections of paraffin or frozen sections, do you know the difference?

The first of these would be in the fixing step . While fixation is carried out in the paraffin sections before the tissue is embedded in it, in the case of frozen sections the tissues are not fixed until after sectioning.

Another of the big differences is the conservation conditions of the tissues . While paraffin sections can be stored at room temperature for long periods of time, frozen sections do not usually remain stable for more than a year, and should always be kept at -80ºC.

Regarding the advantages and disadvantages of each of the techniques, we can highlight the following:

ADVANTAGEDISADVANTAGES
PARAFFIN SECTIONSMaintains the morphological characteristics of the tissuesExcessive fixation time could mask epitopes
FROZEN TISSUE SECTIONSMaintains the enzymatic and antigenic functions of the tissuesCrystal formation could alter tissue structure

Let’s see in more detail what the sample preparation procedure for immunohistochemistry would be in each case.

PARAFFIN SECTIONS

Paraffin-embedded tissue sections are often the choice when intending to preserve tissue samples for long periods of time.

  • Tissue fixation : Before embedding tissues in liquid paraffin, they must undergo a fixation process by perfusion or immersion with the fixative solution followed by dissection.
  • Tissue dehydration : This step is essential because the water is immiscible with paraffin. It is carried out by immersing the tissues in increasing concentrations of alcohol and subsequent incubation with dimethylbenzene (xylene) to remove any residue from it.
  • Embedded in liquid paraffin : The paraffin melts at 60ºC and after embedding the tissues in it, it is allowed to clot again at room temperature. Once solidified, the samples are stored at 4ºC.
  • Cutting sections with a microtome : The paraffin blocks in which the tissues are embedded are cut into ultra-thin sections using a microtome. These sections are fixed on microscope slides that can be stored at room temperature until use.

FROZEN SECTIONS

Sample processing is faster in this case, but the stability of the samples is much less, so they cannot be stored for long periods of time.

On the other hand, in the case of frozen tissues, the sections are usually somewhat thicker, and may lead to poorer resolution under a microscope.

  • Instant Freeze : Tissue is cut into sheets about 3mm thick and frozen by immersion in liquid nitrogen.
  • Cutting sections with a cryostat : When sectioning the tissue it is important that the cryostat is at a temperature between -15ºC and -23ºC since the sections could curl on themselves at too low temperatures, or stick to the blade at temperatures superior.
  • Fixation : In this case, as we have previously commented, the fixation is carried out after freezing and cutting the sections. Typically, the solvent of choice is alcohol, since formaldehyde can cross-react with antigens and mask them.

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