Download NUCLEAR AND CYTOPLASMIC STAINING

 NUCLEAR AND CYTOPLASMIC STAINING Presented by: Jacqueline Brooks MB, HTL, SCT (ASCP) Q IHC, CM IAC Cunningham Pathology [email protected] 1 Housekeeping 2 Cell Morphology 3 Cell Membrane Cell Membrane The outer boundary of the cell. The cell membrane helps control what substances enter or exit the cell. Is composed mainly of protein and lipid molecules. 4 Endoplasmic ReXculum Endoplasmic re2culum The Endoplasmic reXculum is a network of membranes in eukaryoXc cells which helps in control of protein synthesis and cellular organizaXon. A network of interconnected membranes forming sacs and canals. Transports materials within the cell, provides a\achement for ribosomes, and synthesizes lipids. 5 Golgi apparatus Intracellular stack of membrane bounded vesicles in which glycosylation
and packaging of secreted proteins takes place.
6 Ribosomes ParXcles composed of protein and RNA molecules. FuncXon to synthesize proteins. 7 Mitochondria A small intracellular organelle which is responsible for energy
production and cellular respiration.
8 Lysosomes A class of morphologically heterogeneous cytoplasmic particles in
animal and plant tissues latency of these enzymes. The intracellular
functions of lysosomes is characterised by their content of hydrolytic
enzymes and the structure-linked is depend on their lytic potential.
The single unit membrane of the lysosome acts as a barrier between
the enzymes enclosed in the lysosome and the external substrate.
The activity of the enzymes contained in lysosomes is limited or nil
unless the vesicle in which they are enclosed is ruptured. Such
rupture is supposed to be under metabolic (hormonal) control.
9 Peroxisomes LYSOSOMES AND peroxisomes, THE CELL'S "DIGESTIVE SYSTEM" When a white blood cell engulfs a bacterium and destroys it, the white cell's lysosomes do most of ..." 10 Centrosome An organelle located in the cytoplasm of all animal cells and many plants, fungi, and protozoa that controls the polymerizaXon, posiXon, and polar orientaXon of many of the cell's microtubules throughout the cell cycle. 11 Cilia and Flagella The flagella is a tail like appendage that propels the cell. Both the cilia and flagella are a\ached to the outside of the cell. 12 Microfilaments and Microtubules The cytoskeleton (also CSK) is a cellular "scaffolding" or "skeleton" contained within the cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has idenXfied the prokaryoXc cytoskeleton. It has structures such as flagella, cilia and lamellipodia and plays important roles in both intracellular transport (the movement of vesicles and organelles, for example) and cellular division. 13 Nuclear Envelope nuclear envelope The double membrane that separates the nucleoplasm (see nucleus) of a cell from the cytoplasm. The membranes consist of lipid bilayers that are separated by a perinuclear space (or compartment). The outer membrane is conXnuous with the rough endoplasmic reXculum and is structurally and funcXonally disXnct from the inner membrane. The envelope is perforated at intervals by nuclear pores, which provide a channel for the selecXve transfer of water-­‐soluble molecules between the nucleus and the cytoplasm. Each nuclear pore is surrounded by a disc-­‐
shaped structure (nuclear pore complex) consisXng of an octagonal arrangement of eight protein granules. 14 Nucleolus A small dense body (sub organelle) within the nucleus of eukaryoXc cells, visible by phase contrast and interference microscopy in live cells throughout interphase. Contains RNA and protein and is the site of synthesis of ribosomal RNA. The nucleolus surrounds a region of one or more chromosomes (the nucleolar organiser) in which are repeated copies of the DNA coding for ribosomal RNA. 15 ChromaXn Stainable material of interphase nucleus consisXng of nucleic acid and associated histone protein packed into nucleosomes. EuchromaXn is loosely packed and accessible to RNA polymerases, whereas heterochromaXn is highly condensed and probably transcripXonally inacXve. 16 Cancer Atypical adenomatous hyperplasia of the prostate Loss of structural differenXaXon within a cell or group of cells omen with increased capacity for mulXplicaXon, as in a malignant tumor. 17 Metastasis 18 Cause or Causes of Cancer 19 H&E Stain 1. 
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FixaXon Processing Embedding Microtomy Slide Drying DeparaffinizaXon HydraXon Staining (Hematoxylin, differentaXon, bluing, Eosin) DehydraXon Clearing Coverslipping 20 Skin 21 Skin keraXnized squamous epithelium 22 Skin Nucleus open chromaXn 23 STAINING MECHANISMS Most staining reacXons involve both physical and chemical factors. The fat stain is an example of a purely physical stain, with the dye absorbed (soaked up) by, and dissolved in, the lipid. Chemical bonding of the dye to the Xssue: Ionic or electrostaXc bonding occurs when the dye and the substance to be dyed develop different charges and thus become a\racted to each other. Hydrogen bonding occurs when covalently bonded hydrogen is a\racted to atoms that have a strong electronegaXve charge. Covalent bonding occurs when atoms share electrons. Van der Waals forces are caused by the electrostaXc a\racXon of a molecule to the electrons of its neighboring molecules.
24 Skin / RBC’s Blood vessel RBC’s nucleus 25 Prostate Bx High power Nucleus open chromaXn 26 NUCLEAR STAINING (DNA in the nucleus gives it a net negaXve charge) Nuclear staining is not fully understood, but apparently occurs through 2 different mechanisms. • Staining done with the basic (caXonic or posiXvely charged) dyes • Staining done with dyes combined with, or followed by, metal mordants Basophilic – An acidic (anionic, negaXvely charged) substance that is easily stainable with basic (caXonic, posiXvely charged) dyes
27 Prostate Bx low power Open gland Open gland 28 Cytoplasmic Staining •  (proteins are in the cytoplasm giving it a net posiXve charge) •  Acidophilic – A basic (caXonic, posiXvely-­‐charged) substance that is easily stainable with acid dyes. An example is cell cytoplasm, which is readily stainable with the acid (anionic, negaXvely charged) dye eosin. •  If the eosin soluXon is not below pH 6, the eosin will not a\ach to the cytoplasmic proteins because above pH 6, the proteins will have a net negaXve charge. •  IEP (isoelectric point) (the point where the posiXve and negaXve charges are equal) The IEP of proteins is approximately pH 6; below the IEP or below pH 6, the net charge on the nonnuclear proteins is posiXve and the a\racXon is for an anionic dye above the IEP, the net charge is negaXve and the a\racXon is for a caXonic dye. 29 Prostate bx low power normal nuclei Stroma Open gland 30 The Dyes • 
Chromophore-­‐The chemical grouping that bestows the property of color on a compound • 
Chromagen – A benzene derivaXve containing a color-­‐bearing group, or chromophore • 
Auxochrome – The chemical group present in a dye that causes it to bind to certain Xssue elements. This group can develop a charge (+ or -­‐) and thus bind to oppositely-­‐charged groups present in the Xssue. The amino (-­‐NH2) and the carbgoxyl (-­‐COOH) groups are frequently occurring auxochromes. • 
Amphoteric – Describes a substance that is capable of acXng as either a base or an acid, depending on the pH of the soluXon. 31 Breast Xssue Stroma Fat cell nucleus Fat cell 32 Poorly Diff breast ductal CA Stroma Crowded nuclei Cells filling duct 33 Poorly Diff Ductal CA / breast Duct Duct Irregular cells filling duct 34 Factors AffecXng Dye Binding • 
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pH temperature concentraXon salt content fixaXve (formalin react with the NH2 group, because this is the primary group for binding eosin, Xssue fixed in formalin will bind less eosin than when fixed in some of the other soluXons. 35 Gallbladder Lumen RBC’s Glandular cells Smooth muscle 36 Gallbladder RBC’s Glandular Cell Lumen 37 Gallbladder low power Serosal surface Lumen Mucosal surface RBC’s Smooth muscle 38 The Nuclear Dyes • 
Hematoxylin – the most widely used nuclear stain, it is extracted from logwood a tree indigenous to Central America. The freshly cut wood is colorless but becomes dark reddish brown when exposed to atmospheric oxidaXon; the oxidized dye is hematein. SyntheXc hematoxylin is also available but most laboratories sXll use soluXons prepared from the natural product. • 
It is very important to remember that hematoxylin is not a dye; hematein, the oxidaXon product of hematoxylin, is a weak anionic dye. OxidaXon of hematoxylin is necessary and may be achieved naturally by exposing the soluXon to atmospheric oxygen, or by using oxidizing agents such as sodium iodate, mercuric oxide, and potassium permanganate; this oxidaXon process is also called ripening. SoluXons should always contain some un-­‐oxidized hematoxylin because the process of ripening conXnues with atmospheric oxidaXon, and complete oxidaXon or over oxidaXon lead to a breakdown of the soluXon and the loss of good staining. • 
Oxidized hematoxylin (hematein) has li\le affinity for Xssue but becomes a strong dye with a parXcular affinity for nuclei when combined with a metallic mordant. • 
The mordant-­‐dye combinaXon is called a lake, and the most commonly used hematoxylin lakes are combinaXons of hematein with either aluminum or iron. 39 Hematein vs HemaXn •  Hematein – The oxidaXon product of hematoxlin and the acXve staining ingredient in hematoxylin soluXons. Hematein is the acXve dye formed by the acXon of either light and air or a chemical oxidizing agent on hematoxylin. •  HemaXn-­‐ Granular, brownish-­‐black crystalline deposit occurring in Xssue. HemaXn is the term commonly used to denote formalin pigment, formed by the acXon of acidic formaldehyde on blood-­‐rich Xssue, but malarial pigment and acid hemaXn are also hemaXns. 40 Appendix low power Loose ConnecXve Tissue Serosal Surface Mucosal surface Lymphocytes 41 Appendix / lymphocytes Mature lymphocyte Immature lymphocyte Different stages of maturaXon 42 Types of Hematoxylin • 
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Harris Hematoxylin Hematoxylin
Absolute ethyl alcohol Ammonium aluminum sulfate (mordant) DisXlled water Mercuric oxide or sodium iodate (oxidizer)
Delafield Hematoxylin (oxidaXon occurs naturally) SoluXon A Ammonium Aluminum sulfate (mordant) DisXlled water SoluXon B Hematoxylin Alcohol, 95% Glycerol 43 Tonsil / lymphoid Xssue Mature lymphocyte Immature lymphocyte 44 Types of Hematoxylin Mayer Hematoxylin •  Hematoxylin •  DisXlled water •  Sodium iodate (oxidizer) •  Ammonium or potassium aluminum sulfate (mordant) •  Citric acid •  Chloral hydrate Ehrlich Hematoxylin (oxidaXon occurs naturally) •  Hematoxylin •  Alcohol, 95% •  DisXlled water •  Glycerol •  Ammonium or potassium aluminum sulfate (mordant) •  Glacial aceXc acid 45 Umbilical Cord / RBC’s Smooth muscle Red Blood Cells Lumen 46 Types of Hematoxylin Gill Hematoxylin • 
DisXlled water • 
Ethylene glycol • 
Hematoxylin, anhydrous • 
Sodium iodate (oxidizer) • 
Aluminum sulfate (mordant) • 
Glacial aceXc acid • 
Note: Marketed commercially in 3 different strengths and mucin, especially in goblet cells, will be stained by Gill hematoxylin, but not by the other hematoxylin soluXons. Weigert Hematoxylin (Iron Hematoxylin) • 
SoluXon A • 
Ferric chloride, 29% (mordant and oxidizer) • 
DisXlled water • 
Hydrochloric acid, concentrated • 
SoluXon B • 
Hematoxylin • 
Alcohol • 
Note: Not used in the rouXne H&E stain but used as a nuclear stain in many of the non-­‐rouXne techniques because it resists de-­‐colorizaXon in acidic staining soluXons. 47 Placenta / RBC’s Red blood cells or erythrocytes Open chromaXn 48 CelesXne Blue •  Ferric ammonium sulfate (mordant) •  DisXlled water •  Note: CelesXne blue may be subsXtuted for hematoxylin in the H&E procedure and gives idenXcal results. 49 POC lymphocyte AcXve Cell 50 DifferenXaXon Progessively stained slides do not require differenXaXon Regressively stained slides are over stained then excessive stain is removed (differenXaXon) 51 Tonsil / knife mark RBC’s lymphocytes Knife mark 52 Bluing Amer nuclear staining with hematoxylin soluXons and differenXaXon if indicated, the secXons are blued. This is done with soluXons that are weakly alkaline. The change in pH induced by the bluing agent changes the solubility of the dye lake. The aluminum-­‐hematein complex is red and soluble below pH 5; bluing converts the red, soluble complex to a more desirable blue lake that is insoluble in the usual staining soluXons. •  Dilute lithium carbonate • 
Ammonium hydroxide • 
Scot soluXon (magnesium sulfate, sodium bicarbonate, tap water) 53 Plasma Stains The plasma stains are most frequently anionic, or negaXvely charged, dyes that combine with very caXonic, or posiXvely charged, Xssue groups. The basic amino acids, such as arginine, hisXdine, and lysine, are common sites for dye binding. Note: Eosin is the most widely used counter-­‐stain in the rouXne staining of secXons. The best staining with eosin will occur at a pH of approximately 4.6 to 5. Used properly, at least 3 shades of pink can be obtained with eosin alone •  Erythrocytes (deepest shade of pink) •  Collagen •  Cytoplasm of muscle or epithelial cells All the above should stain different intensiXes of pink. Whether the collagen or muscle/
epithelial cells are the intermediate shade of pink is determined by choice of fixaXve, duraXon of fixaXon, heat during processing, stain formulaXon, differenXaXon step and the nature of the collagen itself. 54 Colon Polyp Red blood cells lymphocytes Glandular cells 55 Eosin Eosin Counter-­‐stain •  Eosin Y (1% aqueous soluXon) •  Ethyl alcohol, 95% •  AceXc acid, glacial Eosin-­‐Phloxine B Counter-­‐stain •  Eosin Y (1% aqueous soluXon) •  Phloxine B (1% aqueous soluXon) •  Alcohol, 95% •  AceXc acid, glacial •  Note: Some laboratories prefer an eosin-­‐phloxine B soluXon, because the pink shades are more vivid but must be careful not to over-­‐stain. 56 Colon Polyp / Eosinophil lymphocyte Glandular cells Eosinophil 57 H&E Progressive Stain 1. 
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Xylene, 3 changes 2 minutes each Absolute alcohol 10 dips Alcohol, 95%, 2 changes
10 dips each Tap water
Rinse unXl water runs off evenly Hematoxylin, Mayer
15 minutes Or acidified Harris 1-­‐3 minutes Tap water, 2 changes
10 dips each Ammonia water, 0.25% or
unXl blue Lithium carbonate, 0.5% Tap water, 2 changes
10 dips each Eosin 10-­‐20 dips Or Eosin – phloxine 1-­‐3 minutes Alcohol, 70%
10-­‐15 dips Alcohol, 95%
10-­‐15 dips Absolute alcohol, 3 changes
10-­‐15 dips each Xylene, 3 changes 10-­‐15 dips each Let slides remain in last container of xylene unXl a coverslip is applied. 58 H&E Regressive Stain 1. 
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Xylene, 3 changes
Absolute alcohol
Alcohol, 95%, 2 changes Tap water
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Hematoxylin, Delafield, Ehrlich,
Or Harris without acid 6. Tap water, 2 changes
7. Hydrochloric acid 1% in 70% alcohol
8. Running water
9. Ammonia water, 0.25% Or lithium carbonate, 0.5% 10. Tap water, 2 changes
11. Eosin Or eosin-­‐phloxine
12. Alcohol, 70%
13. Alcohol, 95%
14. Absolute alcohol 3 changes
15. Xylene, 3 changes
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Let slides remain in last container of xylene unXl a coverslip is applied. RESULTS • 
Nuclei
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Erythrocytes and eosinophilic granules
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Cytoplasm and other Xssue elements
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2 minutes each 10 dips 10 dips each rinse unXl water runs off 10-­‐15 minutes evenly 10 dips each 5-­‐10 dips wash well unXl blue 10 dips each 10-­‐20 dips 1-­‐3 minutes 10-­‐15 dips 10-­‐15 dips 10-­‐15 dips each 10-­‐15 dips each Blue Bright pink to red Pink shades 59 Cervical Bx with HPV effect The Koilocyte displays a large perinuclear halo with irregular clear-­‐cut edges (HPV) Koilocyte 60 Cervical Bx High grade Irregular cell with nucleoi Irregular nuclear border 61 Cervical Bx High grade nucleoi Irrgular nuclear border Intranuclear cytoplasmic inclusion Clumpy chromaXn 62 Pap smear low grade with HPV Normal intermediate cell Perinuclear halo 63 Pap/low grade with HPV KeraXnized cytoplasm Perinuclear halo 64 Pap smear high grade HyperchromaXc nuclie Increased N/C raXo 65 Pap smear posiXve adenocarcinoma Adenocarcinoma cells Normal squamous cell 66 Hints to Help Achieve Good H&E Staining • 
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Microscopically check control slide (small intesXne) prior to running paXent samples Do not allow secXons to dry at any point during staining Keep soluXons covered while not in use. Make sure soluXons completely cover slides. If any precipitate is noted at the top of the hematoxylin container, filter the soluXon into a clean dry container.l Develop a rouXne schedule for changing soluXons based on the number of slides stained each day. Record and document. Amer applying the bluing soluXon wash the secXons very well; any carry over of the bluing soluXon will change the pH of the eosin and cytoplasmic staining will be lacking. Do not pass the slides through the dehydraXng soluXons too quickly, because dehydraXng soluXons also serve to differenXate. Keep in mind that the more dilute the alcohol, the more eosin that will be removed. 67 Hints to Help Achieve Good H&E Staining • 
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Tissues that have been fixed for longer than normal may require increased staining Xmes. Staining Xmes also may need to be adjusted according to the fixaXve used, the Xme in hematoxylin may need to be increased amer fixaXon in Helly, Zenker, or B-­‐5 fixaXves, and the Xme in eosin will frequently need to be decreased. If using a xylene subsXtute, the manufacturer’s recommendaXons must be closely followed. Daily check and record the pH of the running tap water. Some tap water may not be acceptable before or amer hematoxylin. Iron, sulfur, and chlorine will produce weak nuclear staining. Chlorine content, which varies seasonally, will also cause staining variability. Highly alkaline or hard water may serve as an excellent bluing agent but may create dark nuclear or background staining. Remember that what appears to be a staining problem is not always a staining problem. If the problem cannot be idenXfied easily as a staining problem, cut and stain secXons from a previous day’s workload in which the staining was excellent. If the staining is sXll excellent on the previous material, then the source of the problem must be in some other area. The duraXon of fixaXon, the use of heat during processing, and the possible carryover of formalin or water into the clearing and infiltraXon reagents are areas that should be examined when an apparent staining problem is proved not to be so. 68 Restoring Tissue Basophilia Staining properXes of markedly over-­‐decalcified bony Xssues cannot be restored, so proper iniXal decalcificaXon is very important. Method I •  Place deparaffinized slides from Xssue overexposed to Bouin soluXon in 5% aqueous lithium carbonate soluXon for 1 hour. •  Wash in running tap water for 10 minutes, and stain using desired method. Method II •  Place deparaffinized slides in a 5% aqueous sodium bicarbonate soluXon for 3 hours (4 hours for Xssues overexposed to Zenker soluXon). •  Wash in tap water for 5 minutes, and stain using desired method. Method III •  Place deparaffinized slides in 5% aqueous periodic acid for 30 minutes. •  Rinse in 3 changes of disXlled water, and stain using desired method. 69 Frozen SecXon Staining • 
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Cut the frozen secXon and fix in 37% to 40% formaldehyde for 20 seconds (I prefer using alcoholic formalin) Rinse the secXon very well in at least 3 changes of tap water (if using alcoholic formalin one water rinse) Stain in Harris hematoxylin with aceXc acid for 1 to 1 ½ minutes Rinse in 2 two changes of tap water Place slide in 0.25% ammonia water or another bluing agent, and leave unXl blue. Rinse in 2 changes of tap water Stain in eosin with 15 to 20 dips or unXl the desired intensity is achieved. Dehydrate with 95% alcohol and absolute alcohol – 10 dips in 2 changes of each alcohol Clear the secXons with xylene – 10 dips in 3 changes Mount with syntheXc resin Results: Nuclei Blue Cytoplasm and other Xssue elements
Shades of pink 70 Notes on Frozen SecXon Staining •  Fix cut secXons immediately; do not allow the slides to air-­‐dry or morphologic preservaXon will be poor •  Can stain with rapid H&E stain or use metachromaXc dyes such as toluidine blue O or polychrome soluXons (purchased commercially) •  SoluXons should be changed on a regular basis depending on usage •  Alcoholic formalin may be used instead of concentrated formaldehyde 71 TROUBLESHOOTING THE H&E STAIN Incomplete Deparaffiniza2on •  Dry secXons properly before beginning deparaffinizaXon; if improper drying is the cause, slides can be treated with absolute alcohol to remove the water, and then re-­‐treated with absolute alcohol to remove the water, and then re-­‐treated with xylene to remove the paraffin; if incomplete drying is severe, the secXons may loosen from the slides •  Allow sufficient Xme in xylene for complete deparaffinizaXon; if this is the cause, return to xylene for a longer Xme •  Avoid contaminated xylene; change the soluXon if necessary •  If the slides have been stained, decolorize and restain Nuclear staining is not crisp •  Fix Xssue specimen completely •  Dehydrate and clear Xssues completely before infiltraXng with paraffin •  Do not use heat on the processor except for the paraffins •  Do not leave Xssue in melted paraffin for a prolonged period •  Dry microscopic slides at the correct temperature (60-­‐70 degrees C) and for the shortest Xme possible that ensures complete drying 72 TroubleshooXng H&E stain Pale Nuclear staining •  Not leaving in hematoxylin long enough •  Staining with over oxidized or depleted hematoxylin •  Over differenXaXng the hematoxylin •  Note: pale nuclei in bone secXons may result from over-­‐decalcificaXon Dark Nuclear Staining •  SecXons lem too long in hematoxylin •  SecXons too thick •  DifferenXaXon step too short Red or Red-­‐Brown Nuclei •  Ensure that secXons are blued properly; it is not possible to over-­‐blue the secXons •  Check oxidaXon status of hematoxylin as given in secXons following “Delafield hematoxylin” 73 TroubleshooXng H&E Stain Pale Cytoplasmic staining •  Check eosin soluXon pH; adjust with aceXc acid if necessary •  Completely remove bluing reagent before transferring the slide to eosin •  Do not allow stained slides to stand in the lower concentraXons of alcohols amer the eosin; the more water in the alcohol, the more eosin that will be removed •  Ensure that secXons are not too thin Dark cytoplasmic staining •  Avoid over concentrated eosin soluXon, especially if phloxine is present; if necessary, dilute the eosin soluXon •  Do not leave secXon in eosin too long •  Allow sufficient Xme in dehydraXng soluXons, especially 70% alcohol, to allow good eosin differenXaXon •  Check secXon for proper thickness 74 Eosin not properly differenXated If 3 shades of eosin are not apparent •  Timely and complete fixaXon •  Good dehydraXon and clearing during processing •  Eosin-­‐stained secXon remains in the lower diluXons of alcohol for proper differenXaXon; adequate Xme in 70% alcohol will give the best differenXaXon of the eosin •  Eosin is at the correct pH 75 TroubleshooXng H&E Stain Blue-­‐Black Precipitate on top of sec2ons •  Filter hematoxylin •  Hazy or Milky water and slides •  When the slides and water turn milky following the rehydraXng alcohols, it indicates the presence of xylene on the slides •  When slides appear hazy or milky in the last xylene used in clearing, it indicates that water is sXll present on the slides and that dehydraXon is not complete. Uneven H&E staining •  May be caused by water or fixaXve in the infiltraXng paraffin, or by contaminaXon of reagents in closed Xssue processors because of equipment malfuncXon or absorpXon of atmospheric water by the dehydraXng alcohols on the open processors. •  Use toluene instead of xylene in areas of high humidity if using open processors; toluene is more water tolerant than xylene •  Check equipment for malfuncXon 76 TroubleshooXng H&E Stain Dark Basophilic staining of nuclei and cytoplasm, especially around 2ssue edges •  Laser and electro-­‐cautery techniques denature macromolecules and produce heat arXfact, generally marked by dark basophilic staining in nuclei and cytoplasm. There is no remedy for this arXfact Poor contrast between nucleus and cytoplasm •  The nucleus is too pale to contrast well with the cytoplasm •  The cytoplasm is over-­‐stained and masks the nuclei •  The nuclear stain is too dark for the cytoplasmic stain •  The cytoplasmic stain is too pale for the nuclear stain 77 Nucleic Acid Stains Feulgen Reac2on • 
For the demonstraXon of DNA • 
Reagents • 
Hydrochloric Acid • 
Schiff Reagent • 
Sulfurous Acid • 
Results: DNA
Reddish purple • 
Cytoplasm Light green Methyl Green-­‐Pyronin Y • 
To differenXate between DNA and RNA • 
Reagents • 
Glacial aceXc acid • 
Sodium Acetate • 
Methyl green dye • 
Results: DNA (green to blue-­‐green), RNA (Red to rose), Goblet cells (mint green, Background (pale pink to colorless), Immunoblast and plasma cell cytoplasm (intense red), Nuclei (green to blue-­‐green) 78 CPT Codes •  Services 88300 through 88309 include accession, examinaXon, and reporXng. 79 THE END Lung Xssue artery GI Xssue 80