Specimen Preparation

Instructions for Preparing Samples for Submission:

Tissue for Paraffin Embedding:
For the best preservation of tissue morphology, the thickness of the tissue placed in fixative should be between 0.2 and 0.5 cm. Sections that are thicker require time for the fixative to permeate to the center, resulting in potential degradation. The volume of fixative should be at least 10 times that of the specimen.

Tissue for Frozen Section:
For the best results, tissue for frozen section histology should not be fixed in a chemical fixative prior to freezing, although there may be specific reasons to do so. Tissue should be gently blotted free of extraneous fluid prior to being placed in a mold, surrounded by a cryoprotectant (e.g. OCT), and being snap frozen. For help with freezing techniques, please contact the histology lab (773.868.4287) or the Perlman lab (773.755.6544). Tissue should be frozen as soon as possible to prevent the detrimental effects of autolysis, including RNA and DNA degradation. Frozen sections can be cut on very small pieces of tissue and care should be taken to not make tissue pieces too large, or they may freeze too slowly, causing ice crystal formation or the development of cracks. If frozen blocks are stored at -80, they should be warmed to the temperature of the cryostat for ease of cutting. After cutting, to preserve the tissue, the cut surface should be covered with OCT and frozen.

Choosing a fixative:
The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues, and for sufficient time to allow the permeation of the fixative into the tissue block. The three most common fixatives are 10% buffered formalin, glutaraldehyde and alcohol. There are many other fixatives that have been reported to incrementally increase the quality of such studies as immunohistochemistry and in situ hybridization. Such fixatives should be used with care, as they also have negative aspects outside these specific tests.
Formalin (10% buffered) is used for all routine surgical pathology tissues, upon which H&E stained slides, special stains, immunohistochemistry, and in situ hybridization is routinely performed. With formalin, tissue is fixed by cross-linkages formed in the proteins between lysine residues. This cross-linkage does not harm the structure of proteins greatly and antigenicity is not lost. For those working with DNA from formalin fixed tissue, the proteins attached to the DNA are likewise cross-linked. Therefore, greater efforts and different techniques may be required to free the DNA for in-situ hybridization. Formalin rapidly penetrates tissues. Tissues can remain in formalin for prolonged periods of time without compromising histology. However, after 24 hours, the longer the tissue is in formalin, the more antigenicity is lost. Prompt processing will stabilize this. For small amounts of formalin, please contact the Perlman lab.

Glutaraldehyde is recommended for fixation of tissues for electron microscopy. Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not ideal for immunoperoxidase staining. However, it fixes very quickly so it is good for electron microscopy. It penetrates very poorly, therefore VERY small pieces of tissue must be put into gluteraldehyde (approximately 1-2mm). However, gluteraldehyde gives the best overall cytoplasmic and nuclear detail of all the fixatives. The standard solution is a 2% buffered glutaraldehyde. If the tissue is left too long in gluteraldehyde, it will become quite hard. Therefore, standard practice is to fix the tissue in gluteraldehyde for no longer than 2 days, and then place the tissue in phosphate buffered saline, in which it can be stored for long periods of time. Electron microscopy is quite expensive and very specialized. It is critical that such studies are carefully planned. Dr. Hector Melin-Aldana is available to help with such planning and can be reached at 773.880.4439 or .

Alcohols, including methanol and ethanol, are protein denaturants and are not used routinely for tissues because they cause too much brittleness and poor morphology. However, they are very good for cytologic smears because they act quickly and give good nuclear detail.

Sample Processing: In the histology laboratory, samples are processed automatically in a machine that starts with formalin, includes alcohol and xylene, and ends with tissue that has been permeated with paraffin. The warm paraffin-permeated tissue is then manually placed in a “block” or cassette that can be sectioned. For tissue that is embedded in alcohol, if notified, the laboratory can skip the initial steps that have the tissue incubating in formalin.

Sample sectioning: Samples are routinely sectioned at 2-5 microns when placed on glass slides. Thicker sections may be desired if mapping structures across many sections. When extracting paraffinized material for DNA or RNA extraction, there are two methods that are utilized. First, unstained slides may be prepared, deparaffinized in xylene, and scraped into containers. Second, unmounted paraffin “scrolls” may be cut and placed into containers for further processing. If RNA is the product of interest, it is important to notify the laboratory so that they can utilize special procedures that prevent cross-contamination.

Distinguishing Orientation:
For some samples, proper orientation is essential. Orientation can be indicated with an eosin dot along with detailed instructions. However, we very strongly recommend contacting Dr. Samanths Gadd(; 773.755.6392) to ensure the most accurate cutting, especially for first-time services. 

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