HTP-NOV-13-STAINING-ARTIFACTS-FAULTS-IN-PROCESSING.pdf

HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 LEGEND IMMUNOHISTOCHEMICAL STAINING PPT...

HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 LEGEND IMMUNOHISTOCHEMICAL STAINING PPT Lecturer Book Combination of immunologic and histochemical stain Black Dark Purple 2 Allow phenotypic markers to be detected and demonstrated Reference book used: Histopathologic Techniques by Gregorios under the microscope STAINING Uses a wide range of polyclonal or monoclonal, fluorescent or PRINCIPLE OF STAINING enzyme-labeled antibodies The process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells. Helps to study and evaluate physical characteristics and structural relationships of tissues and their cells THREE MAJOR GROUPS OF STAINING HISTOLOGICAL STAINING A process whereby tissue components are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of active tissue components Micro-anatomical staining Albert H Coons and his colleagues (Coons et al. 1941, Demonstrates the general relationship of tissues and cells with 1955; Coons and Kaplan 1950) were the first to label differentiation of nucleus and cytoplasm antibodies with a fluorescent dye, and use it to identify antigens in tissue sections Enzymes labels have been introduces such as peroxidase (Nakane and Pierce 1966; Avrameas and uriel 1966) Alkaline phosphatase (Mason and Sammons 1978) Colloidal gold (Faulk and Taylor 1971) label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level MOST COMMON FIXATIVES USED IN IHC 1. 4% paraformaldehyde in 0.1M phosphate buffer 2. 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer 3. PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% HISTOCHEMICAL STAINING lysine in 0.1M phosphate buffer Various components of tissues are studied thru chemical 4. 4% paraformaldehyde with 0.05% glutaraldehyde (TEM reactions that will permit microscopic localization of specific immunohistochemistry) tissue substance Table 1: Basic Protocols of Immunohistochemistry ENZYME HISTOCHEMISTRY Active regent serves as a substrate upon which the enzymes STEP PROTOCOL act Fixation 10% Neutral buffered formalin for 24 hrs in room temperature Final coloration produced is from the substrate rather than the Frozen section: cold acetone for 1 min tissue Embedding and Paraffin embedding sectioning Mostly 4 µm Frozen sections: between 4 µm and 6 µm in thickness Deparaffinization 60°C hot plate and hydration Antigen (or Heat induced epitope retrieval is most widely used epitope) retrieval Blocking Normal sera of same species of secondary antibody or premixed Vary from 30 min to overnight, from 4°C to room temperature Add primary Antibody dilution by protein blocking solution or antibody premixed Ab diluents Appropriate antibody selection and titration Incubate 30-60 min, room temperature Wash (TBS-T) 3x5 min Add secondary - 1  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 antibody FLUORESCENT Incubate 30-60 min, room temperature Useful to visualize multiple antigens simultaneously with Wash 3x5 min, TBS-T multiple fluorophores excited at different wavelength Add substrate 250 µL of 1% DAB, and 250 µL of 0.3% hydrogen (multiplexing) peroxide to 5 mL of PBS, 1-3 mins, room Better, higher-resolution image quality temperature The signal can be amplified by using HRP antibody labels with Wash 3x5 min, DW tyramide-dyes to deposit fluorescent dyes at the site of Counterstain Hematoxylin, 1 min antibody staining TSB-T, Tris-buffered saline & Tween 20; DAB, diaminobenzidine; PBS, More amenable to auto-fluorescence, particularly with phosphate buffered saline; DW, dextrose 5% in distilled water formaldehyde fixation DIRECT VS INDIRECT DETECTION Faster experimental procedure Before choosing your primary, you need to consider whether Requires more expensive imaging equipment you plan to use direct or indirect detection methods. The Fluorophore less stable over time antibody is detected either: POPULAR ENZYMES AND SUBSTRATES/CHROMOGENS FOR IHC DIRECT INDIRECT Through a label that is directly Using a labeled secondary Chromogen/ Mounting Enzyme Color Advantages Disadvantages conjugated to the primary antibody raised against the host Substrate Media antibody species and antibody type and subtype of the primary antibody HRP Diaminobenzidine Brown Organic/ Intense tetrahydrochloride aqueous color; (DAB) permanent Comparison between Monoclonal and Polyclonal Antibodies ANTIBODY ADVANTAGES DISADVANTAGES DAB + Nickel Black Organic/ Intense Endogenous – Great epitope Less sensitivity or reactivity Enhancer aqueous color; peroxidase permanent activity in specificity and to masked epitope in a tissue can lead lower background formalin fixed paraffin to false positive Monoclonal – Better embedded sample staining reproducibility AEC Red Aqueous Intense (3-amino-9-ethylc color; Higher sensitivity/ – Less productivity due arbazole) contrast Recognizing multiple to batch to batch well with epitopes variability blue in Polyclonal double – Higher background due staining to natural antibodies – Limited production AP BCIP/NBT Blue / Organic Intense (5-bromo-4-chloro Black color -3-indolyl-phospha CHROMOGENIC VS FLUORESCENT DETECTION te) / (Nitro blue Your detection methods can be either: tetrazolium) Chromogenic, using secondary antibodies that are Fast Red Red Aqueous Endogenous enzyme-labeled (eg. HRP, AP) AP activity in Fluorescent (Immunofluorescence) using secondary tissue can lead antibodies that are fluorochrome-labeled (eg, FITC, R-PE, to false positive Alexa-Flour) staining CHROMOGENIC Permanent Red Red Organic/ Biotinylated secondary antibodies and streptavidin-HRP can aqueous further amplify the signal in an ABC method. Alternatively, you can use a modern HRP-polymer secondary antibody Some precipitates are photostable (HRP/DAB is very photostable, but HRP/AEC fades in sunlight), potentially allowing storage of the slides for many years Only requires a standard brightfield microscope The enzyme/chromogen precipitate is deposited over a wider area from a fluorescent source, which can affect one’s ability to interpret the results The procedure is generally longer as it includes more incubation and blocking steps than fluorescent methods - however, this isn’t always the case, depending on which amplification system you use Quantification is generally more difficult to enzymatic amplification 2  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 Picture: Immunohistochemical staining of Kidney STAINING REACTIONS DIRECT STAINING The process of giving color to the sections by using aqueous or alcoholic dye solutions (e.g. methylene blue, eosin) ACCENTUATOR Not essential to the chemical union of the tissue and the dye but merely accelerates the staining reaction by increasing the staining power and selectivity of the dye METHODS OF STAINING PROGRESSIVE STAINING Tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until INDIRECT STAINING the desired intensity of color is attained The action of the dye is intensified by adding another agent to No washing or decolorization occurs make the staining possible Differentiation of tissue details relies solely on the selective By itself, the dye may stain only weakly, if at all affinity of the dye REGRESSIVE STAINING Selective removal of excess stain from the tissue in order that a specific substance may be stained distinctly DECOLORIZATION Selective removal of excess stain from the tissue in order that a specific substance may be stained distinctly Basic primary stain ➔ acidic dye as decolorizer Acidic primary stain ➔ alkaline decolorizer Alcohol acts as a differentiator for both basic and acidic dyes MORDANT The term mordant comes from the french word: mordre, “to bite” A substance used to set dyes on tissue sections by forming a coordination complex with the dye which then attaches to the tissue For intensifying stains in cell or tissue preparations 3  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 PROGRESSIVE AND REGRESSIVE HEMATOXYLIN METALLIC IMPREGNATION FORMULATIONS: SIMILARITIES & DIFFERENCES Specific tissue elements are demonstrated, not by stains, but ASPECTS REGRESSIVE PROGRESSIVE by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black Hematoxylin concentration 5gm/L 1-4gm/L deposit on the surface of the tissue or bacteria This is not considered as staining because we use metallic Acetic acid Absent Present ions instead of dye which does not penetrate inside tissues but Rate of uptake Rapid Slow only attaches or forms precipitates on the surface of the tissue. Easy to control No Yes METALLIC IMPREGNATING AGENT Different from a stain in that it is not absorbed by the tissue, but Over staining Yes No is held physically on the surface as a precipitate or as a Differentiation required Yes No reduction product in certain tissue components. The most valuable metals for this purpose are: ○ Gold (gold chloride) METACHROMATIC STAINING ○ Silver (silver nitrate) Solutions should never be exposed to sunlight if explosion is Entails the use of specific dyes that differentiate particular to be avoided, and all unused reagents should be immediately substances by staining them with a color different from the inactivated by sodium chloride or dilute hydrochloric acid stain itself (metachromasia) solution and discarded. Particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules and amyloid STAINS NATURAL STAINS Dyes obtained from natural resources Dye Source Carmine Female Cochineal insect (Coccus cacti) COUNTERSTAINING Application of a different stain to provide contrast and background to the staining of the structural components to be demonstrated Cytoplasmic stains Nuclear stains – Red - eosin Y, eosin B, – Red - neutral red, safranin O, phloxine B carmine, hematoxylin The bug is treated with alum to produce the dye – Yellow -picric acid, orange – Blue - methylene blue, carmine. It is widely used as a powerful G, rose bengal toluidine blue, celestine blue chromatin and nuclear stain for fresh material – Green - light green SF, – The nucleus of a living cell is and smear preparations. lissamine green resistant to vital stains, and Orcein Lichens therefore is not demonstrated VITAL STAINING Selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis) The nucleus of a living cell is resistant to vital stains, and therefore is not demonstrated Intravital staining Supravital staining Done by injecting the dye into Used to stain living cells any part of the animal body immediately after removal from (either intravenous, the living body A vegetable dye which are normally colorless intraperitoneal, or Thin slices of tissue are placed in but when treated with ammonia and exposed to subcutaneous), producing small staining dishes and enough air, produce blue or violet colors specific coloration of certain staining solution is added to cover It is a weak acid that is soluble to alkali and is cells the tissue mainly used for staining elastic fibers 4  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 Saffron Pistils of a flower absolute alcohol, followed by descend grades of alcohol to prevent damage and detachment of sections due to the possible production of diffusion currents Alcohol is replaced with water before actual staining is performed STAINING OF CELLOIDIN SECTIONS Paraffin ribbons containing air bubbles, torn, or adequately infiltrated sections are likely to float from the slide when deparaffinized and stained. they are more firmly attached by coating the slide with dilute (thin) celloidin solutions (collodionization) Cellulose nitrate (celloidin) is soluble in absolute alcohol, hence treatment should be avoided during dehydration and It is a nuclear stain which produces red nuclei clearing of stained sections and is used primarily as a counterstain; may Sections treated with 95% absolute alcohol may be transferred also be used to give a yellow color to collagen to a mixture of equal parts of chloroform, absolute alcohol and xylene, then treated with xylene and mounted in Xam Hematoxylin Heartwood of a log tree PRECAUTIONS IN STAINING 1. Stains on in should be avoided because stains are health hazards per se. Stains may be effectively removed from the skin by prompt topical application of 0.5% acid alcohol, followed by rinsing with tap water. 2. Failure of sections to remain on the slide during staining could have been due to a dirty or oily slide or albumin fixative may be too old. 3. if the section does not stain, the staining solution may be faulty. Impurities found in the dye or in the water solvent will affect the Hematoxylin Stain solubility and intensity of the dye. 4. Failure of staining may be due to paraffin, fixative, or Derived from the core of a Mexican tree known as decalcifying solution that has not been thoroughly washed out Hematoxylin Campechianum. and removed. 5. Stains may be saved and used again for as long as they have Has a powerful nuclear and chromatin staining capacity. not lost their staining properties. 6. If, after staining, sections do not appear clear under the Hematoxylin is not a true basic dye. The active coloring agent is microscope, xylol should be replenished. Hematin which is formed by the oxidation of hematoxylin by a 7. If the tissue is thoroughly adherent to the slide, it can be taken process known as ripening accomplished by exposing the back several times for staining without any danger of peeling substance to air and sunlight, thereby oxidizing hematoxylin. off. RESTAINING OF OLD SECTIONS SYNTHETIC STAINS a. The slide is usually immersed in xylene for 24 hours, or gently Aka “coal tar dyes” since they were originally manufactured heated until the mounting medium begins to bubble from substances that have been taken from coal tar b. The coverslip is removed by lifting it with a dissecting needle Derived from the hydrocarbon benzene (C6H6), and are c. The section is placed in xylene for 30 minutes to remove the collectively known as “aniline dyes” remaining balsam and is brought to water Consists of a chromophore (produce visible colors; coloring d. Place it in a 0.5% potassium permanganate solution for 5-10 property) and a salt-forming auxochrome (gives dye its ability minutes, rinse in tap water and subsequently immerse in 5% to retain color; dyeing property) oxalic acid for 5 minutes ACID DYES e. Wash it in water for another 5 minutes then restain with the Where the active coloring substance is found in the acid appropriate staining technique component. USEFUL STAINS IN HISTOLOGY Picric acid, trichloroacetic acid REGULAR STAINS BASIC DYES HEMATOXYLIN & EOSIN (H & E) STAINING Where the active coloring substance is found in the basic H & E is the most common dye used in the study if histology component Hematoxylin stains acidic structures (nucleic acids, nuclei, Methylene blue rER) blue NEUTRAL DYES Eosin stains basic structures (proteins, membranes) pink Formed by combining aqueous solutions of acid and basic TRICHROME STAIN dyes, capable of staining cytoplasm and nucleus Uses three dyes to differentiate intracellular structures simultaneously and differentially Particularly helpful in highlighting red blood cells within blood Romanowsky dye, Giemsa’s stain vessels STAINING OF PARAFFIN SECTIONS Red keratin and muscle fibers, blue or green collagen and Paraffin wax is poorly permeable to most staining solutions and bone, light red or pink cytoplasm, and dark brown to black cell should therefore be removed from the section prior to staining nuclei Usually done by immersing the paraffin section in a solvent two times, at 1-2 minutes duration each Xylene is not miscible with aqueous solutions and low graded alcohol, and should therefore be subsequently removed with 5  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 IRON-HAEMATOXYLIN Highlights tissue and cells that contain iron, such as muscle & red blood cells SPECIFIC STAINS FOR PROTEOGLYCANS Gomori trichrome Periodic acid-Schiff (PAS) reaction Mallory’s trichrome ○ Also useful for the identification of polysaccharides Masson’s trichrome (Glycogen) Cresyl violet Toluidine blue Methylene blue PERIODIC ACID-SCHIFF (PAS) STAINING PAS reaction is a method to demonstrate glycogen-like substances Periodic acid oxidizes the glycol groups in the glucose residues into aldehyde groups, which then react with Schiff’s reagent, producing an insoluble compound with a reddish purple color Note the blue nuclei, the pink/purple cytoplasm, the red blood cells and the turquoise material that is non-cellular SPECIFIC STAINS FOR NEURONS Nissl ○ A specialized stain for rER of neurons Silver & Gold ○ For fibers & cytoskeletal elements Osmic acid ○ For myelin, a lipid Cresyl violet ○ Proteoglycans NISSL STAINING A specialized stain for rough endoplasmic reticulum in neurons The purplish to blue dot-like structure in the picture is the rER 6  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 SILVER STAINING VERHOFF STAIN FOR ELASTIN Stains elastic fibers dark blue to black RETICULAR STAIN Involves the use of silver impregnation (see picture in the right) ROMANOWSKY STAIN FOR BLOOD CELLS These are useful for highlighting various types of granules present in developing and mature white blood cells Giemsa, Wrights, etc. CRESYL VIOLET Highlights proteoglycans Most commonly used for staining nervous tissue This is a violet/purple stain METACHROMASIA Phenomenon where a certain dye shows structures in a different color from that of the dye. The picture below shows the granules in the cytoplasm of the mast cell in purple color when stained by toluidine blue, a blue dye. ELASTIN Elastin stains elastic fibers black 7  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 TOLUIDINE BLUE Red; typical for staining amyloid fibers Congo red Image is cutaneous amyloid Crystal violet Violet; can stain glia and neurons Pink/orange/red; typical for general Eosin staining when combined with haematoxylin Image is of normal skin Fontana- Black/pink, or red; stains melanin Masson Blue/violet/pink; commonly used in blood Giemsa or bone marrow smears Blue/purple; standard for general staining Haematoxylin when combined with eosin Image is of normal skin Purple/black; can stain mast cells and Luna stain elastin OSMIC ACID STAINING Blue: stains the rough endoplasmic Nissl The effect of osmium tetraoxide is to preserve and stain lipids reticulum in neurons and proteins Red/magenta; used to stain glycogen, Osmic acid serves as both a fixative and a dye basement membranes, reticular fibers, Periodic Acid Schiff cartilage, glycoproteins, glycolipids and (PAS) mucins in tissues. Image shows superficial fungal elements due to candida infection Red Oil 3 Red; used to stain fat emboli Reticulin stain Blue/black; stains reticular fibers ARTIFACTS PRE-HISTOLOGY These are features and structures that have being introduced prior to the collection of the tissues A common example of these include: ink from tattoos and freckles (melanin) in skin samples. POST-HISTOLOGY Artifacts can result from tissue processing. Processing commonly lead to changes like shrinkage, color changes in different tissue types and alterations of the structures in the tissue. Because these are caused in a laboratory, the majority of post histology artifacts can be avoided or removed after being discovered. OIL RED O NON NATURAL OCCURRENCES (ARTIFACTS) This is used to stain lipids a red-orange color in unfixed frozen The main cause of artifacts is poor fixation sections Some tissues usually present artifacts & wrong knowledge has been derived from them Caused by a bad histological technique: ○ Autolysis ○ Poor sampling ○ Shrinkage ○ Folds ○ Stain precipitation and dust ○ Defects in the knife Name of Stain Color and other notes Blue; common mucin stain Alcian blue Image is pretibial myxoedema Purple/black; used to stain beta cells in Aldehyde fuchsin the pancreas Alkaline phosphatase Red/blue; used for endothelial tissue Black; used for neural plaques and Bielschowsky stain tangles 8  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 AUTOLYSIS KNIFE MARKS Suprarenal gland showing autolysis ○ Tubules are not seen anymore EDGE DAMAGE SHRINKAGE AND RUPTURE There are spaces in between tissues We don’t want excessive shrinkage of tissue If we don’t identify it as an artifact, it can be misidentified as atrophy POOR FIXATION The white spaces between the pancreas lobules are artificial Shrinkage that occurs during fixation resulted in this artifact FOLDS DUST 9  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 FAULTS IN PROCESSING Under processed During the processing and cutting of sections, several portion of tissue burst difficulties may be encountered, due to some faults which may on warm contact with have been made in the previous procedures. A good medical water technologist should be alert in taking note of such faults, which Hard spot on the tissue Once embedded in if not immediately corrected, shall cause entire failure in the processing, poor sectioning, and ultimately improper evaluation due to calcium paraffin wax, of the tissue in question. decalcification is impractical; use a FAULTS/PROBLEMS OBSERVED DURING CUTTING base-sledge microtome PROBLEM REASON REMEDY with a knife wedge Surfaces and edges of Re-trim the block Tilt of knife is too great Reduce the tilt the block are not or bevel is not cleared. parallel Hence object is Horizontal surface if Re-adjust and re-orient compressed against the block is not parallel the block the knife edge Sections of Sections fail to to the knife Clamp set screw on Tighten the screw unequal thickness form ribbons Paraffin wax is too Coat horizontal edges knife or block holder is are produced hard of the block with wax of loose lower melting point Blocks are too large Cut block into smaller Knife is tilted too much Reduce the tilt fragment Sections are too thick Re-adjust the thickness Blocks are too hard Soften the blocks in Knife is dull Hone and strop detergent of phenol Sections roll up on Knife is blunt Sharpen the knife Static electricity due to Breathe out or blow cutting so that they Tilt of knife is too great Reduce the tilt low atmospheric gently on the block and adhere and get humidity knife to break up static Knife edge is dirty Clean the knife edge Sections adhere to electricity, or boil water broken against the the knife or other knife edge in the room to increase parts of the humidity Blunt or dull spot on Adjust the knife so that microtome the knife, producing the knife will present a Knife edge is dirty Clean the knife edge and irregular knife uniformly sharp edge Knife edge is dull Sharpen the knife Ribbon is curved, edge to the block, or Knife tilt is too great Reduce the tilt crooked, or sharpen Nicks or damage on Sharpen the knife uneven instead of Edges of the block are Re-trim the block the knife edge straight not parallel but round Dirty embedding Re-embed in freshly Ribbon split or or wedge shaped filtered wax if lengthwise vertical Knife is not parallel to Re-adjust the knife and necessary scratches are the block block Knife edge is dirty Clean the knife edge seen on sections Knife is blunt or dull Re-sharpen the knife with xylene Paraffin block is warm Cool the block on ice Tilt of the knife is too Reduce the tilt and soft water until firm great Knife edge is coated Clean the knife edge Knife tilt is too great Reduce the tilt Sections are Sections are lifted Knife is dull Sharpen the knife with paraffin compressed, from the knife on Paraffin is too soft or Cool the block on ice Sections are too thin Readjust the thickness wrinkled, or upstrokes room temperature is water until firm of the section jammed warm Microtome set crew is Tighten the screw loose Tilt of the knife is too Increase the tilt Tilt of knife is too Reduce the tilt small, paraffin block is Resistance is felt vertical therefore compressed on the lower part Sections are Bevel knife is lost due Re-sharpen, using a against the base of the of the section squashed (width of to incorrect sharpening knife back or automatic knife towards the end during cutting sections is less knife sharpener of the stroke than that of the block) Horizontal or Knife edge vibrates Treat with phenol Bubble or dirt is Re-embed in freshly parallel lines or due to hardness of during processing or formed in the filtered wax if furrows across the tissue collodionize embedding medium necessary section (“chatters”) Tilt of knife is too great Reduce the tilt A hole is formed in Tissue is not Re-process the tissue are seen the section processed properly Knife is blunt Sharpen the knife Section cut is and will not form a Knife is not clamped Adjust the knife so that sometimes thin, section (esp. If center properly knife edge will present sometimes thick is raw) a uniformly sharp edge 10  HISTOPATHOLOGY & PATHOLOGY PREBOARD EXAM REVIEW HIRAYA | RMT 2024 to the block, or sharpen Knife or block holder is Tighten adjusting and loose locking screws Knife tilt is too small Increase the tilt that block is compressed by bevel and section is not cut Knife makes a Tilt of knife is too Readjust the tilt hard metallic slanted or too big scraping or ringing Tissue is too hard Take fresh block sound on treated with phenol backstroke, when during processing section is cut Knife blade is too thin Change the knife Frozen tissue Freezing is not Refreeze the tissue crumbles and adequate block comes off the block holder when cut Frozen tissue Tissue is frozen too Warm the tissue with chips into much fingers fragments when cut Top and bottom edges Adjust the block holder of block are not to make the block Ribbons are parallel to edge of edges parallel to the crooked blade/sides of block knife are not perpendicular to the blade Sections are too Wrong micrometer Microtome needs thick setting calibration Clearing agent not Block is trimmed down completely removed nearest to the tissue. due to insufficient Remaining wax is On trimming, impregnation melted on embedding tissue smells of oven and paraffin clearing agent impregnation is repeated, changing the paraffin at least once before embedding Insufficient clearing Repeat clearing; if the Tissue is opaque, object has already section cutting is been embedded, difficult due to prolong clearing up to presence of 12 hours. Then alcohol re-embed Insufficient Repeat the whole Tissue shrinks dehydration, therefore procedure away from wax insufficient clearing when trimmed and impregnation Contaminated wax Re-embed in freshly On trimming, wax Block is not cooled filtered wax if appears crystalline rapidly enough necessary Paraffin block, Insufficient paraffin Repeat paraffin after cooling, is impregnation, then moist and re-embed crumbles 11