HISTOPATH

TRIMMING - Certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater
- Once the tissues have been embedded and the wax has solidified, the wax block is removed affinity for basic dyes, while basic constituents (e.g. cytoplasm) take more of the acid stains.
from the mold, the identification number is noted and the excess wax is cut off from the - Many dyes, however, require the use of a mordant - a chemical compound that reacts with
block to expose the tissue surface in preparation for actual cutting the stain to form an insoluble, colored precipitate on the tissue and make the staining
- The sides, top and bottom of the tissue block are trimmed until perfectly level and all sides reaction possible.
are parallel, almost to the edge of the tissue
- An old knife or blade may be used for this procedure, but it must still be relatively sharp to STAINING OF PARAFFIN SECTIONS
avoid damage to the tissue - This is usually done by immersing the paraffin section in a solvent (e.g. xylene) two times, at
- Coarse facing is done on the microtome at approximately 30 microns at a time until the 1-2 minutes duration each, for sections up to 10 micron thick.
entire tissue surface is exposed. Care should be taken to avoid removing too much tissue in - The alcohol is then finally replaced with water before actual staining of section is
this step performed. Such procedure is the exact reverse of impregnation and may be summed up by
- Fine trimming may be done by either setting the thickness adjuster at 15 mm or by the phrase "Sections to Water".
advancing the block using the coarse feed mechanism. - After the section is cut and mounted on the slide, it is drained and dried thoroughly to
- The surface block is then trimmed away until the entire tissue surface has been partly ensure that all moisture between the section and slide has evaporated, and that the section
exposed. is firmly attached to the slide.
- The block is advanced into the knife and cutting is continued until complete sections come - After staining, the section is again dehydrated with increasing grades of alcohol and cleared
out of the block and a regular cutting rhythm is maintained with two changes of xylene to prepare the section for mounting, since most mountants are
- Sections are cut between 4-6µ in thickness for routine histologic procedures, after the block miscible in xylene.
has been fixed and secured to the block holder
- The actual thickness of the first couple of sections in a ribbon may be thicker than indicated
HISTOLOGICAL STAINING
because of thermal expansion when cutting a cold paraffin block.
- Histological staining is the process whereby the tissue constituents and general relationship
- Using the microtome handle, try to cut in a slow and consistent manner - don’t start and
between cell and tissue are demonstrated in sections by direct interaction with a dye or
stop while the blade is cutting a block as this may produce horizontal lines across the block
staining solution, producing coloration of the active tissue component.
and the sections (and very slight changes in thickness).
- It is important to remember that the colors of stains are not the real color of a particular
- Sectioning is generally improved when the specimen and the wax are well matched in
tissue, and that a structure that appears as one color using one stain, may be a quite
hardness. It is for this reason that most paraffin blocks must be cold when sections are cut.
different color using another stain.
- Cold wax provides better support for the harder elements in a specimen allowing thinner
sections to be obtained. Place the blocks on a cold plate or a cold wet surface for a few
METHODS OF STAINING
minutes (such as the surface of melting ice)
DIRECT STAINING (POSITIVE STAINING)
- Sections are removed in ribbons of ten to allow easy location of serial sections. The sections
- the process of giving color to the sections by using
are then floated out on a water bath set at 45-50°C, approximately 6-10°C lower than the
aqueous or alcoh olic dye solutions.
melting point of the wax used for embedding the tissue.
- In simple (or direct) staining only one dye is used, which is
- This is to flatten the sections and prepare them for mounting onto the slides. Folds and
washed away after 30–60 seconds, prior to drying and
creases may be removed by stretching the sections gently with a pair of dissecting needles
examination.
or forceps
- The molecules that make up basic dyes have a positive
- A section is selected for staining and picked up onto a clean slide in a vertical position. The
charge.
slide is immersed in the water bath in a near vertical position as close as possible to the
- This is important because the cell wall and cytoplasm of bacterial cells have a negative
section.
charge.
- When the slide touches the section, it is lifted vertically out of water and drained.
- The positively charged dye is attracted to the negatively charged cells, enhancing the ability
- Flotation should expand the section to its original dimensions and ensure that it is
of the stain to stick to and color the cells.
completely flat. The temperature will need to be 5 - 9 ˚C below the melting point of the
- Methylene blue is a classic example of a simple stain. This blue stain will color all cells blue,
wax.
making them stand out against the bright background of the light microscope.

FAULTS/PROBLEMS OBSERVED DURING SECTION-CUTTING

INDIRECT STAINING (NEGATIVE STAINING)
FAULT CAUSE REMEDY
- The process whereby the action of the dye is
Tear or scratch across Jagged knife edge Sharpen the knife
the section or splitting Dirt or hair on knife edge. Clean the knife intensified by adding another agent or a
of ribbon. MORDANT
Tear or scratch across Calcium, Carbon, or Suture etc., Examine block under  MORDANT – serves as a link or bridge
part of section in the tissue or wax magnifying glass. If calcium is between the tissue and the dye, to make the
present, decalcify block. staining reaction possible.
Remove suture from the tissue - The mordant combines with a dye to form a
with scalpel point if dust is in
colored "lake", which in turn combines with the tissue to form a "tissuemordant-dye-
wax
Re-embed complex" that is rendered insoluble in ordinary aqueous and alcoholic solvents.
Holes in the section Air bubbles in the tissue or wax Re-embed - This allows subsequent counterstaining and dehydration to be carried out easily
A piece of hard material in Remove hard material if - A mordant may be applied to the tissue before the stain, or it may be included as part of the
tissue possible staining technique, or it may be added to the dye solution itself.
A soft piece of tissue in block Reprocess specimen - Examples of mordants are potassium alum with hematoxylin in Ehrlich's hematoxylin, and
iron in Weigert's hematoxylin.
Section shows thin and A loose knife Tighten knife and/or block
thick horizontal lines A loose block Sharpen the knife
(chatters) A blunt knife Soften the tissue if possible or ACCENTUATOR
Extremely hard tissue embed in harden wax - is not essential to the chemical union of the
Section cut thick and Knife tilt is too great and is Adjust tilt tissue and the dye. It does not participate in
thin alternative compressing the block the staining reaction, but merely accelerates
the reaction.
- Examples are potassium hydroxide in Loeffler's
STAINING methylene blue and phenol in carbol thionine
- Staining is the process whereby tissue components are made visible in microscopic sections and carbol fuchsin.
by direct interaction with a dye or staining solution.
- Most cells are colorless and transparent, and therefore histological sections have to be
stained in some way to make the cells visible. The same is true of components of the PROGRESSIVE STAINING
extracellular matrix. - Progressive staining is the process whereby tissue elements are stained in a definite
- The main reason why cells are stained is to enhance contrast and visualization of the cell or sequence, and the staining solution is applied for specific periods of time or until the
certain cellular components under a microscope. desired intensity of coloring of the different tissue elements is attained.
- Once the dye is taken up by the tissue, it is not washed or decolorized. The differentiation
HISTOLOGIC STAIN or distinction of tissue detail relies solely on the selective affinity of the dye for different
- Purified form of a coloring agent or crude dye that is generally applied in an aqueous cellular elements.
solution.
- The actual staining process may involve immersing the sample (before or after fixation and REGRESSIVE STAINING
mounting) in dye solution.
 - With this technique, the tissue is first overstained to obliterate the cellular details, and the 5. Thionine
excess stain is removed or decolorized from unwanted parts of the tissue, until the desired 6. Toluidine blue
intensity of color is obtained.
- Routine Hematoxylin and Eosin (H&E) staining is the most common method utilized for HEMATOXYLIN AND EOSIN (H & E) STAINING
microanatomical studies of tissues, using the regressive staining which consists of - Hematoxylin and Eosin (H&E) staining is the corner stone of tissue-based diagnosis. The
overstaining the nuclei, followed by removal of superfluous and excessive color of the tissue process stains thin tissue sections so that pathologists can visualize tissue morphology.
constituent by acid differentiation. - The process uses a hematoxylin dye to stain cell nuclei (and other parts) blue and an eosin
dye to stain other structures pink or red.
DIFFERENTIATION (DECOLORIZATION) - Hematoxylin binds strongly to acids and consequently binds to nuclear DNA and stains
- selective removal of excess stain from the tissue during regressive staining in order that a nuclei blue.
specific substance may be stained distinctly from the surrounding tissues. - This detail is required for tissue-based diagnosis, particularly in the detection and
- A staining procedure that differentiates or distinguishes between types of bacteria is classification of infection, cancer or metabolic disease.
termed as a differential staining technique - It is also generally done before any additional staining techniques, because histology with
H&E can confirm the basic tissue type and help to localize the lesion. (The term lesion is
DIFFERENTIAL STAINING used by pathologists to indicate any area of damage, infection, inflammation, tumor,
- uses more than one chemical stain to better differentiate between various microorganisms necrosis or otherwise abnormal tissue.)
or structures/cellular components of a single organism.
- This is usually done by washing the section in simple solution (e.g. water or alcohol), or by ROUTINE H&E STAINING IN PARAFFIN EMBEDDED SECTION (REGRESSIVE STAINING)
the use of acids and oxidizing agents. FIXATION: Most fixatives can be used except osmic acid solutions which inhibit hematoxylin.
- One commonly recognizable use of differential staining is the Gram stain. Gram staining
uses two dyes: Crystal violet and Fuchsin or Safranin (the counterstain) to differentiate PROCEDURE:
between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and 1. Clear paraffin embedded sections in first xylene bath for 3 minutes.
Gram-negative bacteria. 2. Transfer to second xylene bath for 2 to 3 minutes.
3. Immerse in first bath of absolute ethyl alcohol for 2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 or 2 minutes.
5. Rinse in running water for 1 minute.
6. Stain with Harris alum hematoxylin for 5 minutes (Ehrlich's
7. hematoxylin requires 15-30 minutes).
8. Wash in running tap water to remove excess stain.
9. Differentiate in 1% acid-alcohol (1ml concentrated HCl to 99 ml. of
10. 80% ethyl alcohol) for 10-30 sec. monitoring the changes in color
11. microscopically until only the nuclei are stained.
12. Rinse in tap water.
13. Blue in ammonia water (average of 5 minutes) or 1% aqueous
14. lithium carbonate until the sections appear blue (about 30 seconds).
15. Wash in running water for 5 minutes.
16. Counterstain with 5% aqueous eosin for 5 minutes. If alcoholic
17. eosin is used, the time can be reduced to 30 seconds or 1 minute.
18. If aqueous eosin is used, wash and differentiate in tap water under
METACHROMATIC STAINING 19. microscope control until the nuclei appear sharp blue to blue black and
- entails the use of specific dyes which differentiate particular substances by staining them 20. the rest of the tissue appear in shades of pink. If alcoholic solution is
with a color that is different from that of the stain itself (metachromasia). 21. used, differentiate with 70% alcohol.
- This is particularly employed for staining cartilage, connective tissues, epithelial mucins, 22. Dehydrate, clear and mount.
mast cell granules, and amyloid.
- At its simplest, the actual staining process may involve immersing the sample (before or FROZEN SECTION STAINING
after fixation and mounting) in dye solution, followed by rinsing and observation. - Frozen sections mounted on the slides may be stained as in paraffin sections although the
duration of staining is usually shorter.
METALLIC IMPREGNATION - Frozen sections may be stained by picking up sections on albuminized slides and drying
- a process where specific tissue elements are demonstrated, not by stains, but by colorless them quickly or by simple direct staining on a wet slide with an eye dropper
solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, - The following staining methods are commonly employed for frozen sections, the choice
usually black deposit on the surface of the tissue or bacteria. depending upon the personal preference of the pathologist and the type of tissue section to
- Ammoniacal silver, for example, is reduced by argentaffin cells (e.g. in melanin and be stained
intestinal glands), forming black deposits seen under the microscope. 1. Hematoxylin-Eosin method
2. Thionine method
VITAL STAINING 3. Polychrome Methylene Blue method
- the selective staining of living cell constituents, demonstrating cytoplasmic structures by 4. Alcoholic Pinacyanol method (used also for supravital staining of mitochondria and primarily
phagocytosis of the dye particle (cytoplasmic phagocytosis), or by staining of pre-existing for color sensitization in photography)
cellular components (true vital staining), as in the staining of mitochondria by Janus green
- The nucleus of a living cell is resistant to vital stains, and therefore is not demonstrated. In H & E STAINING OF FROZEN SECTIONS FOR RAPID DIAGNOSIS (PROGRESSIVE STAINING)
fact, demonstration of nuclear structures during vital staining suggests permeability of the 1. Orient section in the block and freeze with liquid nitrogen.
membrane of the dye, signifying the death of the cell. 2. Cut cryostat sections at 5-10 micron.
3. Mount sections on to albuminized slides and dip in 10% formalin to
INTRAVITAL STAINING 4. fix.
- Intravital staining of living cells is done by injecting the dye into any part of the animal body 5. Rinse rapidly in water.
(either intravenous, intraperitoneal or subcutaneous), producing specific coloration of 6. Stain with Harris hematoxylin for 30-45 seconds.
certain cells, particularly those of the reticulo-endothelial system. 7. Rinse in tap water.
- Common dyes used are lithium, carmine and India ink. 8. Blue in ammonia water for 5 seconds.
9. Rinse in tap water.
10. Counterstain with 5% aqueous eosin or 1% alcohol eosin for one
SUPRAVITAL STAINING
11. minute.
- Supravital staining is a method of staining used in microscopy to examine living cells that
12. Rinse in tap water.
have been removed from an organism. It differs from intravital staining, which is done by
13. Dehydrate in increasing concentrations of alcohol.
injecting or otherwise introducing the stain into the body
14. Clear with xylene.
- Those that enter and stain living cells are called supravital stains (e.g. New Methylene Blue
15. Mount with cover slide.
and Brilliant Cresyl Blue for reticulocyte staining)
- Note that many stains may be used in both living and fixed cells. Thin slices of tissues are
placed in small staining dishes and enough staining solution is added to cover the tissue. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
- Histochemical staining is the process whereby various constituents of tissues are studied
thru chemical reactions that will permit microscopic localization of a specific tissue
MOST COMMONLY USED SUPRAVITAL STAINS:
substance.
1. Neutral red – probably the best vital dye.
- Chemical ions such as calcium, molecules such as bile pigments, and biopolymers such as
2. Janus green – especially recommended for mitochondria.
cellulose, DNA and specific enzymes are among the tissue components that can be
3. Trypan blue – one gram of dye is dissolved in 100 ml. of sterile distilled water to be used
identified using histochemical staining techniques.
immediately; it is dangerous to allow the suspension to stand for more than one hour,
- Examples of such type of stains are Perl's Prussian blue reaction for hemoglobin, and
because it is likely to become toxic to the cell.
Periodic Acid Schiff staining for carbohydrates.
4. Nile blue
IMMUNOHISTOCHEMICAL (IHC) STAINING FORMULA:
- A combination of immunologic and histochemical techniques using a wide range of - 1% gelatin 5 ml
polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and - 2% formaldehyde 5 ml
demonstrate tissue antigens (e.g., proteins) and phenotypic markers under the microscope. - Coat the slides with the above mixture. Allow coated slides to dry at 37°C for one hour
- Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as or overnight before use.
those found in cancerous tumors, in the localization of biomarkers and differentially
expressed proteins in different parts of a biological tissue, and in the detection of specific 5. POLY-L-LYSIN E
molecular markers that are characteristic of particular cellular events such as proliferation - This aqueous detergent can be purchased as a 0.1% solution which is further diluted
or cell death (apoptosis). 1:10 with distilled water (final dilution to 0.01%) prior to use.
- Immunohistochemical staining techniques are used to label defined antigens with - Sections are coated with this diluted poly-L-lysine and allowed to dry. This is widely
monoclonal and polyclonal antibodies. used as a section adhesive in immunohistochemistry.
- The current recommendation for immunohistochemical techniques is a maximum of 4% - PolyL-lysine coated slides must be used within a few days after they are prepared, since
neutral buffered formaldehyde solution and, for some antibodies, fixation time can be up to its effectiveness as an adhesive slowly decreases in time.
a maximum of 48 h.
- Microwave-based fixation of tissue in formaldehyde may have an adverse effect on
6. APES (3-AMINOPROPYLTHRIETHOXYSILANE)
immunohistochemical staining.
- APES-coated slides are very useful in cytology, particularly for cytospin preparations of
proteinaceous or bloody material. The slides are dipped in 2% APES in acetone, drained,
ADHESIVES AND MOUNTING MEDIA dipped in acetone, drained again, and finally dipped in distilled water.
- Mounting is the last step in tissue processing that results in a permanent histological - They are then placed upright in a rack and allowed to dry. APES-coated slides are better
preparation suitable for microscopy, after adhesion of the sections on to the slide and than poly-L-lysine coated slides because they can be stored for a long time without
appropriate staining of the tissue. losing their adhesiveness.
- Bubbles may also be removed by pulling the ribbon very gently across the long edge of a
glass slide held below the section in the water bath.
MOUNTING MEDIUM
- After draining, the sections are fixed to the slides. This can be done either by leaving the
- If an unmounted stained section is seen in the microscope, differences in light refraction
slides in a 37°C incubator overnight, by placing the slides in a wax oven at 56° to 60°C for 2
between the glass slides and the tissue components, may lead to difference in length
hours, or by drying the slides on a hot plate at 45° to 55°C for 30 to 45 minutes.
dispersion; hence, very little microscopic detail can usually be appreciated.
- Another alternative to drying is by the use of adhesives. To promote adhesion of sections,
- A mounting medium is usually a syrupy fluid applied between the section and the coverslip
adhesives may be spread thinly and evenly on a clean grease-free slide which is then gently
after staining, setting the section firmly, preventing the movement of the coverslip.
approximated to the end of the ribbon, and drawn upwards in a near vertical motion.
- A mounting medium is usually a syrupy fluid applied between the section and the coverslip
- An adhesive is a substance which can be smeared on to the slides so that the sections stick
after staining, setting the section firmly, preventing the movement of the coverslip.
well to the slides.
- Mounting media are often chosen for a specific refractive index (R.I.), which can enhance
- Slides must always be grease- and dust- free and stored and handled correctly.
specimen details or make them invisible.
- If staining is to include antigen retrieval (IHC), enzyme pretreatment for in-situ hybridization
- A clean, dry cover glass is placed on the edge of the slide and gradually inclined downward until
(ISH), or prolonged incubation steps, charged slides or an adhesive must be used.
it touches the mounting medium and gently pressed on to the slide while the mounting
medium quickly spreads through the whole area of the section.
CERTAIN INSTANCES WHEN SECTIONS MAY FLOAT FROM THE SLIDE: - Excessive mounting medium will cause it to ooze out of the sides of the cover glass, and should
a) For urgent cryostat sections to be submitted for immunocytochemistry be carefully wiped with a fine cloth moistened with xylene.
b) For central nervous system tissues - Excessive blotting, on the other hand, will dry up the section, causing shrinkage and cracking of
c) For tissues containing blood clot the specimen.
d) For tissues which have been decalcified - If the section has to be remounted, the cover glass may be removed by soaking in xylene.
e) When sections are to be subjected to high temperatures
CHARACTERISTICS OF A GOOD MOUNTING MEDIUM:
ALBUMIN 1. It should be colorless and transparent.
- most commonly use adhesive is 2. It should be freely miscible with xylene and toluene.
- is prepared by mixing equal parts of glycerin, distilled water and white of eggs, then 3. It should not dry to a non-stick consistency and harden relatively quickly.
filtered through coarse filter paper and a crystal of Thymol is added. 4. It should protect the section from physical damage and chemical activity (oxidation and
changes in pH).
POLY-L-LYSINE 5. It should be resistant to contamination (particularly microorganism growth).
- a favorite adhesive 6. It should not cause shrinkage and distortion of tissues.
- can be bought as a 0.1 % solution and further diluted (1 in 10 with distilled water) when 7. It should not leach out any stain or affect staining.
ready to use. 8. It should not change in color or pH.
9. It should be compatible with the adhesive in use.
AMINOPROPYLTRIETHOXYSILANE (APES) 10. It should set without crystallizing, cracking or shrinking (or otherwise deform the tissue
- is a better section adhesive and coated slides can be stored for a long time. Slides are being mounted) and not react with, leach or induce fading in stains and reaction products
dipped in 2% APES in acetone drained then dipped in acetone, drained again and finally (including those from enzyme histochemical, hybridization, and immunohistochemical
dipped in distilled water. procedures).

ADHESIVES
1. MAYER'S EGG ALBUMIN MOUNTING MEDIA MAY BE DIVIDED INTO TWO MAIN GROUPS:
FORMULA: a. Aqueous Media
- Egg White 50 cc. b. Resinous Media
- Glycerin 50 cc.
- Filter and add about 100 mg. crystals of thymol to prevent the growth of molds. AQUEOUS MOUNTING MEDIA
- Mayer's egg albumin is the most commonly used because it is very easy to make, is - Aqueous mounting medium are used for mounting sections from distilled water when the
convenient, and is relatively inexpensive. A drop of Mayer's egg albumin is usually stains would be decolorized or removed by alcohol and xylene as would be the case with
smeared into the clean glass slide before sections are oriented. most of the fat stains (Sudan methods) or for metachromatic staining of amyloid.
- They are usually made up of gelatin, glycerin jelly or gum arabic (to solidify the medium),
2. DRIED ALBUMIN – dried, and stored in 70% alcohol until it is ready for staining. glycerol (to prevent cracking and drying of the preparation), sugar (to increase the
FORMULA: refractive-index), and a preservative solution.
- Dried albumin 5 gm
- Sodium chloride 5 gm WATER
- Dissolve in 100 cc. of Distilled Water and add crystals of thymol. - has a low refractive index, is moderately transparent and evaporates easily, hence is good
only for temporary mounting. Also, water does not allow tissues to be examined under the
3. GELATIN (1%) oil immersion lens. In a wet mount, the specimen is suspended in a drop of liquid (usually
FORMULA: water) located between slide and cover glass.
- Gelatin 1 gm
- Distilled water 100 ml GLYCERIN
- Glycerol 15 ml - may also be used as a preservative. It has a high index of refraction and provides greater
- Phenol Crystal 2 gm visibility if slightly diluted with water (for moist sections). This is a very suitable semi-
- Adding up to 30 ml of 1% aqueous gelatin to the water in a floating out bath and mixing permanent mounting medium with a refractive index of 1.46, sets quite hard, and will keep
it well is a most convenient alternative to direct coating of slides sections mounted for years, especially if sealed on the edges with paraffin wax. It is miscible
with water, is inexpensive, and is non-poisonous.
4. GELATIN-FORMALDEHYDE MIXTURE
RESINOUS MOUNTING MEDIA
- used for preparations that have been dehydrated and cleared in xylene or toluene, and are
recommended for majority of staining methods. They may be divided into natural and
synthetic resins. The most important synthetic resins are used for embedding undecalcified
bones, and for electron microscopy

CANADA BALSAM (REFRACTIVE INDEX 1.524)

- Canada Balsam is a natural resin extracted from the Canadian tree, Abus Balsamea, usually
dissolved in xylene in an incubator at 37°C or paraffin oven at 58 °C, and filtered, obtaining
the desired consistency by controlled evaporation of the solvent.
- It is a transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard
consistency without granulation. However, it darkens slightly with age and slowly becomes
acid because it oxidizes xylene, thereby causing gradual fading of many stains.
- Canada balsam is recommended for whole mounts and for thick sections because it does
not shrink much. It sets hard without granulation; it is, however, quite expensive.
- As Canada Balsam does not mix with water, mounting in it implies the use of a sequence of
dehydration, starting with low grade alcohols, followed by high grade alcohols, absolute
alcohol, mixed clearing agents plus alcohol, clearing agents, clearing agents mixed with
xylene, pure xylene, and balsam dissolved in xylene.

DPX - (DIBUTYL PHTHALATE AND XYLENE) (REFRACTIVE INDEX 1.532)

- This is a resinous medium recommended for small tissue sections but not for whole mounts
because of shrinkage produced on drying; hence, it should be used in excess amounts.
- It is a colorless, neutral medium in which most standard stains are well preserved. It is
prepared by dissolving the common plastic, polystyrene, in a suitable hydrocarbon solvent
(usually xylene).
- It tends to set quickly and, in doing so, often retract from the edge of the coverslip. It has a
greater advantage over Canada balsam in that slides can be cleaned of excess mountant
simply by stripping it off after cutting around the edge of coverslip.

XAM (REFRACTIVE INDEX 1.52)

- Xam is a synthetic resin mixture in xylene, available in a pale yellow or colorless solution. It
dries quickly without retraction, and preserves stains well. Sections are quickly mounted
from xylene.

CLARITE (REFRACTIVE INDEX 1.544)

- Clarite (or Clarite X) is a synthetic resin which is soluble in xylene (it is used as a 60%
solution in xylene), and is generally preferred over D.P.X. Other recommended synthetic
mounting media include Permount (made by Fisher Scientific), H.S.R. (Harleco Synthetic
Resin), and Clearmount (Gurr).

COVER SLIPPING
- The stained section on the slide must be covered with a thin piece plastic or glass to protect
the tissue from being scratched, to provide better optical quality for viewing under the
microscope, and to preserve the tissue section for years to come.
- The stained slide is taken through a series of alcohol solutions to remove the water, then
through clearing agents to a point at which a permanent resinous substance beneath the
glass coverslip, or a plastic film, can be placed over the section.
- Bubbles under the coverslip may form when the mounting media is too thin, and as it dries
air is sucked in under the coverslip. Contamination of clearing agents or cover slipping
media may also produce a bubbled appearance under the microscope

RINGING
- Ringing is the process of sealing the margins of the cover-slip to prevent the escape of fluid
or semi-fluid mounts and evaporation of mountant, to fix the coverslip in place, and to
prevent sticking of the slides upon storage.
- The term “ringing” originated because round coverslips were initially used and the coating
applied in the form of a circle or “ring.”