Trimming tissues for histology

After the tissues you have collected have been fixed for a sufficient amount of time (see section on fixatives for different guidelines), they must be trimmed before being delivered to the histology laboratory for embedding. Proper trimming of the tissues will ensure that they are presented on the slide in an orientation that will allow appropriate interpretation of their cellular structure by the pathologist. Presentation is critical when trying to identify any variation from "normal" or any pathologic change present in the tissue being viewed.

Before the tissues can be trimmed, it is necessary to decalcify the bones. This is accomplished by an overnight soak in a hydrochloric acid decalcifying solution such as Cal-EX (Fisher, Pittsburgh, PA). After decalcification, bones may be immediately trimmed after an initial rinse with water, but must be continually rinsed in running water for at least 3 or 4 hours before being processed by the histology laboratory. Failure to thoroughly rinse decalcified tissues may result in inadequate staining of the tissue sections. Optimization of the times required for decalcification and washing should be customized for every laboratory.

After any necessary decalcification has been completed, trimming of all tissues may commence. Use a cork board, similar to the one used during your necropsy, as the base on which to trim your tissues. Each tissue, as it is trimmed, should be placed into a labeled and numbered histology cassette (OmniSette Tissue Cassettes, Fisher Scientific, Pittsburgh, PA). Use a #2 pencil, solvent-resistant marker (Histo-Prep Pen, Fisher Scientific), or mechanical labeling machine to label the front and/or side of each cassette. Indicate the mouse's accession number and, if there will be more than one cassette of tissue per mouse, number each cassette for that animal in sequential order. Sequential numbering will assist you in determining whether all tissues sent to histology are returned. Furthermore, knowing which tissue went into which cassette will help identify specific tissues (left vs. right, liver vs. kidney, tumor 1 vs. tumor 2, etc.). Some laboratories always put the same tissues into the same numbered cassette for standardization. Cassettes containing tissues that were fixed and not decalcified should be placed into a container of 70% ethanol, while those containing decalcified tissues not previously rinsed should be placed in water. After a thorough rinsing, decalcified tissues may be placed into ethanol with the other tissues and delivered to the histology laboratory for processing and embedding. Any remaining tissues not submitted for embedding may be stored in 70% ethanol for future use, if necessary.

When handling fixed tissues, it is still important to be gentle. Soft parenchymal organs, such as the liver, brain, lungs, kidneys, etc., remain delicate after fixation and should be manipulated using a pair of wide wooden forceps or a similar tool. Trimming is best done with a single-edge razor blade, which, if sharp, can allow a clean, concise cut with minimal damage to the tissue. Residual chemicals on the tissues can dull a razor blade rapidly, so it is important to change to a new blade frequently while trimming.

All tissues should be trimmed to a thickness no greater than the depth of the histology cassette (4 to 5 mm). If it is necessary to present a particular facet of a trimmed tissue to the pathologist, this must be indicated by marking the side of the tissue one does not want presented. A blue colored pencil works well (Venus col-erase, #1276 blue, Eberhard Faber, Inc., Lewisburg, TN), because the blue pigment will not wash off in alcohol and will clearly indicate the desired orientation to the histologist who will be embedding the tissue. Be sure to mark the side not to be sectioned, because the blue pigment will contaminate the field in a histological section, making it difficult for a pathologist to correctly interpret the tissue being presented. Small, related tissues of similar densities may be placed together in the histocassettes (i.e., kidneys with spleen and/or liver, reproductive tract tissues together, etc.). The heart, lungs, brain, bones and any possible tumors found should each go into a cassette by themselves. The following is a description of trimming technique for each organ. As mentioned previously, any remaining tissue not submitted for embedding may be saved for future use in 70% ethanol.

Large and small intestine—Intestines need no extra trimming at this point, because they should have been prepared before fixation. Each segment should be carefully removed from its backing, if rolled, and placed individually in a histocas-sette.

Stomach and cecum—Both these organs should be cut in half longitudinally. The stomach should be cut to present both the esophageal and duodenal openings. The cecum should be cut to show both the ileocecal junction and the ampula of the colon. Submit the half of each that best shows the desired features. If space allows, the cecum and stomach may be submitted in the same histocassette. If your study is focused on lesions of the cecum or stomach, it may be important to submit both halves of one or both of these organs. If this is the case, each organ should be placed in its own cassette.

Liver (with gallbladder)—Cross sections of only the left lateral lobe of the liver and the medial lobe with the gall bladder may be sent to the histology laboratory unless any pathologic changes are obvious only on the accessory lobes. Lay the left lateral lobe out flat on your trimming surface and cut a crosswise section from anywhere near the center of the lobe (Figure 5.9). Trimming the medial lobes must be a bit more precise, because they must be cross-sectioned in such a way as to include a portion of the gall bladder in the section. This is usually accomplished by cutting across the medial lobes just below the juncture where the lobes separate, then just above that juncture, where the falciform and teres ligaments hold the two lobes together. The first cut should reveal a portion of the gall bladder. Mark the opposing side with blue pencil and place both this section and the left lateral lobe section in the histocassette.

Mouse Liver Histology
FIGURE 5.9 Example of trimming sites for a mouse liver. (Drawn by Ingrid K. Sundberg.)

Kidneys—The left kidney should be cut longitudinally down the center and should include a segment of the adrenal gland which was left attached to the kidney at the time of fixation (Figure 5.10). The right kidney will be identified by a small transverse incision, if handled properly at the time of fixation. It should be presented in a lateral cross section cut through the central area near the pelvis. Use the blue pencil to mark the side of this section farthest from the pelvis, because it is important to present the area nearest the center of the kidney to the pathologist. The kidney sections may be placed in the histocassette with the liver sections.

Spleen and pancreas—Unless you have collected the pancreas separately for focused study, the spleen and pancreas should have been fixed as a unit. Trim them together in cross section at any point along the length of the spleen. This section of the spleen and pancreas may be submitted in the same cassette as the liver and kidneys, if space allows.

Lungs—The lungs are collected with the heart and thymus as a unit at the time of necropsy but are submitted separately for histological studies. Using a pair of wooden forceps, gently push apart the heart and lungs so that the lungs lay out flat. Cut a longitudinal section from the center of the lobes of the lung on each side. The individual lobes will separate, but should all be collected and placed in the same histological cassette. There is generally no need to separately identify each lobe of the lung.

Heart and thymus—Carefully remove any remaining lung or tracheal tissue from around the heart, being certain not to separate the thymus from the heart. Place the heart on its base and begin your cut at the heart's apex. Angle your cut so that it bisects each of the four chambers of the heart (Figure 5.11). In some instances, the coronary artery may be visible on the epicardium of the fixed heart. Making a cut along the line of this vein will often bisect the chambers properly. It may take time to become familiar with the external features of the heart before you can regularly make this cut correctly. If space allows, both halves of the heart may be submitted. If only one half is submitted, make sure that it contains a portion of the thymus.

FIGURE 5.10 Anatomic location of both kidneys and adrenal glands. Right kidney is trimmed transversely, while left is cut longitudinally so that histologic sections can be identified. Lower panels illustrate features of cut sections. (Drawn by Ingrid K. Sundberg.)

FIGURE 5.11 The heart is trimmed lengthwise to include all four chambers. (Drawn by Ingrid K. Sundberg.)

Salivary gland—Trim the base of the salivary gland to present a clean-cut edge. Make your second cut approximately 4 mm from the first cut. Mark the face of the second cut with blue pencil and place the cross section in a histocassette.

Trachea and thyroid/parathyroid—Cut a cross section of the trachea at the point where the thyroid and parathyroid glands are attached. This is located in an area 1 to 2 millimeters below the epiglottis. A V-shaped cut made between the molars at the base of the tongue will free the soft tissue from the bone, or the entire lower jaw may be decalcified. If decalcified, the cut to reveal the trachea and surrounding glands may be made straight across the base of the mandible so that the section includes the molars and bones of the mandible.

Lymph nodes—There are many lymph nodes located throughout the body, but it may not be necessary to submit all of these for processing unless involved in the disease process. Representative nodes from several areas may be chosen (i.e., Mesenteric, axial, inguinal, cervical). If not enlarged, lymph nodes may be submitted whole, after the surrounding fat has been removed. Severely enlarged lymph nodes must be cut in cross section.

Urinary bladder—The urinary bladder should be fixed as a unit with the reproductive organs. A lengthwise cut made down the center of the urinary bladder should also include the uterine body, vagina, and cervix in the female, and the penis and prepuce in the male (Figure 5.13). After this first cut is made, trim the opposite side of one half of your tissue to the appropriate width to fit in your cassette (~4 to 5 mm), cutting off the uterine horn or seminal vesicle and any excess adipose tissue. Mark this side with blue pencil and place the section in a histocassette.

FIGURE 5.12 Female reproductive tract. Dotted lines indicate where to cut tissue for histologic processing. (Drawn by Ingrid K. Sundberg.)

Reproductive organs—Female: Cut one ovary away from either uterine horn, trim away excess fat and place the entire ovary and its associated uterine tube in a histocassette (Figure 5.12). Into the same cassette, place a cross section of one of the uterine horns. Trimming the uterine body, vagina, and cervix is discussed above along with the urinary bladder.

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FIGURE 5.13 Male reproductive tract. Dotted lines indicate sampling sites for histology. (Drawn by Ingrid K. Sundberg.)

Male: Separate the testes from the index card on which they were fixed. The testes and epididymis should be slightly flattened and lie in the same plane on the surface that was attached to the card. Mark the rounded side of the testis with blue pencil and place into a histocassette. Into the same cassette, place a cross section from one side of the seminal vesicles (Figure 5.13). The penis and prepuce are discussed above along with the urinary bladder.

In both male and female, all reproductive organs, as well as the urinary bladder, may be submitted in the same cassette.

Clitoral/preputial glands—The clitoral gland of the female mouse is normally quite small and is fixed in a segment of inguinal fascia and fat. This segment should be cut in cross section and embedded on edge. It may be submitted in the same cassette as the reproductive organs. The preputial gland of the male mouse is larger and is collected individually. It should be cut in cross section as well and sent with the reproductive organs.

Brain—The brain is submitted to the histology laboratory cut in three cross sections rostral to caudal (Figure 5.14). The first cut should be made through the cerebrum, ~1 to 2 mm from the olfactory lobes. The second cut through the cerebrum, 2 to 4 mm from the first, will create the first section. This first section should present a view of the central portion of both cerebral hemispheres, so mark the face of the first cut with blue pencil. The third cut should be made 2 to 4 mm from the second cut, just to the cerebral side of the confluence of sinuses. This will create the second cross section, of which the face created by the second cut should be marked with blue pencil. A fourth cut should be made at the transverse sinus to separate the remaining portions of the cerebral hemispheres from the cerebellum, after which a fifth cut is made, which approximately bisects the cerebellum laterally to create the third section. On this section, the cut face closest to the cerebrum should be marked with blue pencil. The brain should be submitted in a cassette by itself.

Tongue—The tongue should be cut longitudinally down the midline. It is only necessary to submit half of the tongue to histology.

Legs (long bones)—One each of the fore and hind legs should be cut longitudinally to show the long bones and major joints of each leg (Figures 5.15 and 5.16). Excess fat should be trimmed away and on the hind legs it may be necessary to trim

FIGURE 5.14 Trimming sites marked for sectioning a mouse brain. (Drawn by Ingrid K. Sundberg.)

away some of the bulk of the muscle in order to fit the section properly into the cassette. Feet should also be separated from the leg, and will be submitted separately. The longitudinal cut should be made using the major joint of each leg as a reference to bisect the long bones. Choose the half of each leg that best shows the desired view of the bones and place it in its own histocassette.

Feet—One each of the front and back feet should be cut longitudinally to show the skin, foot pads and bones of the feet. The front foot should be cut so two toes are present on each half. The back foot should be cut directly through the middle toe on that foot. Each foot is placed into a separate histocassette. Both halves of each foot may be sent to the histology laboratory.

FIGURE 5.14 Trimming sites marked for sectioning a mouse brain. (Drawn by Ingrid K. Sundberg.)

away some of the bulk of the muscle in order to fit the section properly into the cassette. Feet should also be separated from the leg, and will be submitted separately. The longitudinal cut should be made using the major joint of each leg as a reference to bisect the long bones. Choose the half of each leg that best shows the desired view of the bones and place it in its own histocassette.

Feet—One each of the front and back feet should be cut longitudinally to show the skin, foot pads and bones of the feet. The front foot should be cut so two toes are present on each half. The back foot should be cut directly through the middle toe on that foot. Each foot is placed into a separate histocassette. Both halves of each foot may be sent to the histology laboratory.

FIGURE 5.15 Front leg. (A) Trim site with skin removed. (B) Decalcified limb cut lengthwise to expose joints. (Drawn by Ingrid K. Sundberg.)

FIGURE 5.16 Rear leg. (A) Preliminary preparation of leg includes removal of skin and amputation of distal segment as indicated. (B) Decalcified limb is cut lengthwise to expose joints. (Drawn by Ingrid K. Sundberg.)

Spinal column—The spinal column should be trimmed to present both lateral and longitudinal sections of the thoracic and lumbar regions. First, cut the spine laterally between the thirteenth thoracic and first lumbar vertebrae (just below the 13th rib). Next, cut a lateral section 4 to 5 mm wide, from the distal end of each section. Bisect the remaining long segments longitudinally, placing the best half of each into a separate cassette with its related lateral section (Figure 5.17).

FIGURE 5.17 Longitudinal section of decalcified spinal column. (Drawn by Ingrid K. Sundberg.)

FIGURE 5.17 Longitudinal section of decalcified spinal column. (Drawn by Ingrid K. Sundberg.)

Skull—The skull should be cut into three cross sections, similar to the brain (Figure 5.18). The sections should show the eyes, nasal passages, ear canals and pituitary gland. The first cut should be made through the posterior edge of the pituitary, identified as a whitish mass located in the approximate area between the occipital bone and basiphenoid bone on the inner surface of the skull. This is the crucial cut and should present a view both of the pituitary and the middle ear. The second cut may be made 4 to 5 mm anterior to the first cut to create the first section. The third cut should be made through the posterior edge of the visible portion of the eyes, with the following cut made just anterior to the eyes. The third section should present a view of the sinuses, and may be cut from the approximate center of the remaining portion of the snout. Each section should be marked with blue pencil on its anterior surface. All sections of the skull may be submitted in the same cassette.

FIGURE 5.18 Three sections are cut in the decalcified skull. This exposes: (A) the nasal cavity, (B) eyes and associated glands, and (C) inner, middle, and external ear, as well as the pituitary gland. (Drawn by Ingrid K. Sundberg.)

Skin—Trim portions of the dorsal and ventral skin longitudinally in the direction of the hair growth, into pieces approximately 0.3 x 1.0 cm. Cut 2 to 3 sections of both dorsal and ventral skin in this manner and mark one long edge with blue pencil to indicate that the pieces should be embedded on the opposite edge. Depending on the focus of your study, you may also want to cut a piece of skin approximately 0.7 x 0.7 cm square to be submitted horizontally, haired side down, to view the hair follicles in horizontal section. Mark the underside of the section with blue pencil. Tail skin should be trimmed in the same orientation as the longer pieces of dorsal and ventral skin. Cut the section from an area that was not handled as the tail skin was removed from the bone, and mark one long edge with blue pencil. Eyelids may be presented by making a cut bisecting the lids of both eyes, then making a second cut just posterior or anterior to the corners of the eyelids so that the upper and lower eyelids remain attached to one another (Figure 5.19). Mark the face of the second cut with blue pencil. A section of the muzzle skin may be obtained by making a cut approximately 3 to 4 mm in from the front edge of the muzzle (Figure 5.19). Mark the outer, uncut, edge with blue pencil. Trim a section out of one ear by first cutting one of the ears in half lengthwise and then cutting one of the halves completely off from the scalp (Figure 5.19). Lay this half flat on your cutting surface and make a second cut parallel to the first to obtain a section similar in size and shape to that of the dorsal and ventral skin sections. Put a blue mark on one of the long sides of the section of ear. The various skin sections may be combined into histocassettes. However, you should combine the same pieces every time, so if you have questions later about whether you are looking at dorsal or ventral skin you can figure things out by looking at the other types of skin included. We combine the long sections of dorsal skin with the sections of ear and tail skin into one cassette. The long sections of ventral skin go with the sections of eyelid and muzzle in a second cassette, and we put each of the square sections of dorsal and ventral skin into individual cassettes. Each cassette is also labeled with a "D" or "V" to aid in identification.

FIGURE 5.19 Skin of the head trimmed to study (A) muzzle and vibrissae, (B) eyelids, cilia, Meibomian gland, and conjunctiva, and (C) pinna of the ear. (Drawn by Ingrid K. Sundberg.)

Hematoxylin & Eosin (H&E) stain should be requested on all tissues sent to the histology laboratory. Special stains may be requested on specific tissues if this is necessary to verify any suspected pathologic changes not sufficiently disclosed by the H&E stain. Protocols for various stains, what they stain, and what colors they stain are subjects of various histology and pathology text books.1721

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FIGURE 5.19 Skin of the head trimmed to study (A) muzzle and vibrissae, (B) eyelids, cilia, Meibomian gland, and conjunctiva, and (C) pinna of the ear. (Drawn by Ingrid K. Sundberg.)

Hematoxylin & Eosin (H&E) stain should be requested on all tissues sent to the histology laboratory. Special stains may be requested on specific tissues if this is necessary to verify any suspected pathologic changes not sufficiently disclosed by the H&E stain. Protocols for various stains, what they stain, and what colors they stain are subjects of various histology and pathology text books.1721

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    What is trimming in tissue process?
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