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H&E staining

Nuclear and cytoplasmic staining in the histology lab.

Two majors parts of a cell Nucleus and Cytoplasm
Resting nucleus is typically in what phase? Interphase
The nucleus appears to be what color when being stained with the standard H&E stain? Dark blue to purple
Electron microscopes are able to view subcellular particles which include Nuclear membrane, nuclear pores, nucleolous, chromatin, plasmalemma, mitochondria, ribosomes, endoplasmic reticulum, Golgi apparatus, centriole, and lysosomes
Most likely the avenues of communication between the cytoplasm and the nucleus Nuclear pores
Usually intensely basophilic but with a good H&E stain it is acidophilic Nucleolous
Produces most of the ribosomal RNA Nucleolous
Heterochromatin Stainable
Euchromatin Non stainable
Consists of chromosomes or DNA and attached protein Chromatin
Intensely basophilic Heterochromatin
This type of tissue will exhibit the most intense nuclear staining of all cells because heterochromatin dominates. Lymphocytes
Euchromatin is predominantly present in this type of tissue and will not stain well with heme. Neuronal nuclei
Energy producing powerhouses of the cell Mitochondria
Site of protein synthesis Ribosomes
Some are on the endoplasmic reticulum while others are located in the cytoplasm Ribosomes
Responsible for the blue color in the cytoplasm Ribosomes
Packaging apparatus of the cell Golgi apparatus
Responsible for spindle formation in cell division Centriole
Occur in pairs Centrioles
Part of the bodies defense mechanism Lysosomes
Aid in the digestion of food taken into the cell Lysosomes
Long lived or permanent cells such as neurons, cardiac muscle, and hepatocyte accumulate a large amount of residual bodies. it is referred to as Lipofuscin (wear and tear pigment)
Exogenous materials Carotene, dusts, and minerals
Melanin and hemoglobin breakdown endogenous pigments
Attraction for minute particles from the surrounding solution, by the surface of certain tissue components; the dye is then bound to the tissue primarily by ionic, covalent, or hydrogen bonds Adsorbtion
Ionic bonding Different charges that become attracted to one another
hydrogen bonding Covalently bonded hydrogen is attracted to atoms that have a strong electronegative charge.
Hydrogen frequently bonds to what elements Oxygen and Nitrogen
Covalent bonding When atoms share electrons
Elements that typically form covalent bonds Carbon, hydrogen, and oxygen
Van der Waals forces Caused by electrostatic attraction of a molecule for the electrons of its neighboring molecules. These are weak physical forces that are effective over only very short distances.
Nuclear staining is performed in what two ways? 1. Done with basic (cationic or positively charged) dyes 2. Done with dyes combined with or followed by metal mordants
Staining performed with a basic dye depends on what? Presence of the nucleic acids (DNA and RNA) to form dye salt type unions
Staining performed by combining the dye with or followed by a mordant depends on what? Nucleic acids have been removed (decalcified tissue) and also may occur in tissue that is not negatively charged.
Non nuclear staining is primarily cause by proteins or charged groups on the side chains of amino acids constituting the proteins
Proteins that can be positive or negative Amphoteric
Eosin must be kept below pH of 6 or else the -COO group of eosin recombines with hydrogen and the result is the free acid, uncharged form of eosin. Non specific staining will occur.
A group that confers the property of color Chromophore
if reduction occurs to a chromopohore what happen? Color is lost
A benzene derivative containing chromophoreic groups Chromogen
An ionizing group that links firmly to the tissue Auxochrome
Amino (NH2) Auxochrome
Chromophore C=O, C=S, C=N, N=N, N=O,NO2
Picric acid is anionic or an acid dye because of the 1 auxochrome group
Anionic auxochromes Sulfonic, carboxyl, and hydroxyl groups
Cationic dyes Chloride salts
Basic dyes Crystal violet and safarin
Acid dyes Orange G and picric acid
Amphoteric dyes Hematein and lithium carminate
Factors that affect dye binding 1.pH 2. Temp 3. Concentration 4. Salts 5. Fixative used
Tissue binds less with Eosin when this type of fixative is used Formalin
Tissue binds less with Heme when this type of fixative is used Potassium dichromate
Tissue will lose its nuclear staining properties when stained with these types of fixatives Zenker, Bouin, unbuffered formalin, (all acidic fixatives)
Types of fixatives that increase tissue basophilia or the uptake of cationic or positively charged dyes Formaldehyde, mercuric chloride, and osmium tetroxide
increases the binding of anionic or negatively charged dyes Picric acid
Metals that act as a link between the tissue and the dye Mordants
Ways to differentiate 1. When basic or cationic dyes are used differentiate by weak acid 2. Excess mordant 3. oxidizers
Potassium permanganate and chromium trioxide Oxidizers
An example of what stain uses excess mordant to differentiate Iron heme stains
Aluminum hemes can be differentiated with Dilute hydrochloric acid
Eosin can be differentiated with Dilute solution of Ammonium hydroxide
Hematein Oxidized heme
Hematoxylin can be oxidized by air, light, sodium iodate, mercuric oxide, and potassium permanganate
Harris Heme Hematoxylin Absolute Alcohol Ammounium aluminum sulfate Distilled water Mercuric Oxide
Original formula of Harris Heme used to use mercuric oxide. What do they use now? Sodium Iodate
Harris Hematoxylin mordant and oxidizer Mordant: Aluminum Oxidizer: Sodium Iodate
Harris hematoxylin is used Progressively and acidified
Delafield hematoxylin Solution A Ammonium aluminum sulfate Distilled water Solution B Hematoxylin 95% alcohol Glycerol
Delafield hematoxylin mordant and oxidizer Mordant: Aluminum Oxidizer: light and air
Glycerol does what in the Delafield hematoxylin Stabilizes the solution against over oxidation and prevents rapid evaporation
To check for over oxidation of Delafield heme Drop a few drops into a container of water. If it is blue-black it can still be used. If the solution turns into a red or red brown then it will be over oxidized.
How to check for under oxidation of Delafield heme Drop some heme onto filter paper if the drop has a purple edge it is ready to use. If the purple edge is absent then it is under oxidized.
Delafield heme is used Regressively
Mayer heme hematoxylin Distilled water Sodium Iodate Ammonium or potassium aluminum sulfate Citric Acid Chloral hydrate
Mayer heme mordant and oxidizer Mordant: Aluminum Oxidizer: Sodium Iodate
Citric Acid and Chloral hydrate do what in the Mayer heme solution? Adjust the pH and helps prevent the scum and precipitates
Recommended heme for immunoperoxidase techniques when 3-amino-9-ethylcarbazole is used Mayer heme is used because it does not contain alcohol and will not dissolve the reaction product.
Very difficult to over stain with this heme and produces very crisp nuclear staining. (Progressive) Mayer heme
Ehrlich heme Hematoxylin Alcohol 95% Distilled water Glycerol Ammonium or potassium aluminum sulfate Glacial acetic acid
Ehrlich heme mordant and oxidizer Mordant: Aluminum Oxidizer: natural or chemical can be used
Ehrlich heme More commonly used regressively but can be used progressively
Gill heme Distilled water Ethylene glycol hematoxylin, anhydrous Sodium Iodate Aluminum sulfate Glacial acetic acid
Ethylene glycol does what in the Gill heme? Prevents the formation of surface precipitate
Mordant and oxidizer of Gill heme Mordant: Aluminum sulfate Oxidizer: Sodium iodate
Gill 2 and 3 are used for staining tissue
Gill 3 is most concentrated
Can be used for staining glycol methacrylate sections Gill 3
Gill 1 and 2 are used Progressively
What structure is stained in tissue when using Gill heme Goblet cells in Mucin
Less prone to surface precipitate Mayer and Gill
Scott Solution Magnesium sulfate Sodium bicarbonate Tap water
Scott solution Changes the pH and allows the slides to blue (change color) Also stabilizes the stain.
Weakly alkaline solutions Lithium carbonate, ammonium hydroxide, or Scott solution
Weigert heme Ferric chloride Distilled water hydrochloric acid Hematoxylin 95% alcohol
Resists decolonization in acidic staining solutions Weigert heme
Weigert mordant and oxidizer Mordant and oxidizer: Ferric Chloride
Substitute for Weigert heme Gallein iron heme
how many shade of pink should be obstained when staining with Eosin Three (Erythrocytes, collagen, and muscle)
The more dilute alcohol the more _ will be removed Eosin
Longer the tissue is fixed the A The longer it may need to stain
Time may need to be increased in Heme and decreased in Eosin when fixed in Helly, Zenker, or B-5
Restoring tissue basophilia: If over exposed to Bouin Place in 5% aqueous lithium carbonate for 1 hour
Restoring tissue basophilia: If over exposed to Zenker Place in 5% aqueous sodium bicarbonate for 3 hours
Restoring tissue basophilia: Method III 5% periodic acid for 30 minutes
Tissue basophilia loss may result from -wet tissue too long in Bouin, Zenker, or unbuffered formalin -overdecalification of bone
Best heme to use to stain nuclear chromatin in specimens that have been over exposed to unbuffered formalin and/ or an acid-decalcifying agent Weigert iron heme
White spots may be seen in tissue sections incomplete deparaffinization
Ways to correct incomplete deparaffinization -Ensuring the sections are dried properly -Allow enough time in xylene -Ensure xylene is not contaminated -If slides are stained then decolorize and restain
When distinct chromatin pattern cant be seen (Smudgy or muddy nuclear detail) Nuclear staining is not crisp
Most frequent cause of nuclear staining not being crisp Overfixation
Other causes of nuclear staining not being crisp Too much heat during processing or drying of the microscopic sections
Smudgy nuclei can be prevented by -Complete fixation of specimens -Tissues should be dehydrated and cleared completely -No heat on processor -Tissue should not remain in melted paraffin for long -Dryer is correct temp
Pale nuclear staining 1. Not leaving the slides in heme long enough 2. Staining with overoxidized or depleted heme 3. Overdifferentiating the heme
Over decalcified specimen Pale nuclear staining
Pale nuclear staining can be corrected by -Ensuring slides are left in heme long enough -Ensure heme is not overoxidized -Ensuring differentiation step is properly times -restaining section -use of fixative
Dark nuclear staining most likely causes 1. Sections left too long in heme 2. Sections too thick 3. Differentiation step too short
Dark nuclear staining can be corrected or prevented by -Ensuring sections are thing -Decolorize the sections and restain -Decrease the time sections remain in heme -Increasing the time of differentiation
Red or brown nuclei is caused by Either the heme is breaking down or blueing step was not properly done
Ways to prevent or correct red/red-brown nuclei -Ensure sections are blued properly -Check the oxidation of the heme
Pale cytoplasmic staining may result from pH of the Eosin being >5. It may also occur from sections being too thin or being left too long in dehydrating solution
Pale cytoplasmic staing can be prevented or corrected from -Checking the pH of Eosin -Ensure the blueing reagent is removed before transferring the slides to eosin -Ensure that stained slides are not allowed to stand in low concentrations of alcohols after eosin -Ensure sections are not too thin
Dark cytoplasmic staining can be corrected/prevented by: -Ensure the eosin solution is not too concentrated -Ensure sections are not left too long in Eosin -Ensure time in dehydration solutions allow good differentiation -Check section for proper thickness
Eosin not properly differentiated may be prevented or corrected by -Ensuring timely and complete fixation -Ensure good dehydration and clearing during processing -Ensure eosin stained sections get proper differentiation -Ensure eosin is correct pH
Blue-black precipitate on top of sections Caused from the metallic sheen that is formed on top of heme solutions (Filter to correct)
Water and slides turn milky when slides are placed in water following alcohol during deparaffination if after rehydrating alcohols then it indicates the presence of xylene. Back the slides up and change the alcohols. The take slides from alcohol to water. Water should be clear
Slides are hazy or milky in last xylene before applying cover glass Water is still present. Back up the slides and change the alcohol and xylene. Then rehydrate and clear sections.
Uneven H&E staining Caused by water or fixative infiltrating paraffin caused by contamination of reagents in closed tissue processors because of equipment malfunction or absorbtion of atmospheric water by dehydrating alcohols on the open processors
Uneven H&E staining cant be corrected but can be prevented by -Change from xylene to toluene in areas of high humidity if using an open processor -Have equipment checked for malfunctions
Dark basophilic staining of nuclei and cytoplasm (Especially around the edges Laser and electrocautery techniques (No remedy)
Poor contrast between nuclei and cytoplasm causes 1. nucleus is too pale to contrast with the cytoplasm 2. the cytoplasm is overstained and masks nuclei 3. the nuclear stain is too dark for the cytoplasmic stain 4. the cytoplasmic stain is too pale for the cytoplasmic stain
Poor contrast between the nuclei and the cytoplasm can be prevented or corrected by: -Determine which stain is the problem -Check the pH -monitor water pH
DNA is found in the nucleus
RNA is found in the nucleolous and ribosomes
Feulgen reaction purpose Demonstration of DNA
Feuglen reaction principle Based on the mild hydrolysis of DNA by hydrochloric acid. Removes purine bases but leaves sugars and phosphates of DNA intact. Hydrolysis generates an aldehyde group that can be demonstrated with Schiff.
Feuglen reaction fixative Anything but Bouins
Feuglen reaction results DNA: Reddish purple Cytoplasm: Light green
Methyl green-pyronin Y purpose Demonstrates DNA and RNA
Primarily used to identify plasma cells and immunoblasts in tissue Methyl green-pyronin Y
Methyl gree-pyronin Y principle DNA stains with green, while RNA is colored red with pyronin. Differential staining caused from differing degrees of polymerization. Methyl green is bound by more highly polymerized DNA.
Methyl green pyronin Y fixative 10% NBF is preferred. B-5, Helly, or Zenker is fine
Methyl green pyronin Y results DNA: Green to blue-green RNA: rose Goblet cells: Mint green Background: Pale pink to colorless Immunoblast and plasma cell cytoplasm: Intense red Nuclei: Green to blue-green
Polychromatic stains A compound dye or dye mixture that contains components of different colors
A process in which a dye forms other dyes spontaneously Polychroming
Romanowsky type stain Giemsa
May Grunwald Giemsa stain Purpose To permit differentiation of cells present in hematopoietic tissue. Also used to stain certain microorganisms
May Grunwald Giemsa stain Principle "neutral" dyes combine the basic dye methylene blue and the acid dye eosin give a wide color range when staining blood smears. Because of impurities present in dye solution.
May Grunwald Giemsa stain Fixative Zenker or B-5 preferred, 10% NBF is fine
May Grunwald Giemsa control Spleen
May Grunwald Giemsa Results Nuclei: Blue Cytoplasm: Shades of pink, gray, blue Bacteria: Blue
Tissue fixed in aldehydes will have increased basophilia
What are two types of mounting medium? Resinous and Aqueous
Resinous media tends to be natural
Examples of resinous media Canada balsam and gum dammar
Natural resins dissolve in what? Xylene
Natural resins Take a long time to harden, turn yellow over time, and tend to cause fading in stains over time due to their acidity.
Most resinous media are dissolved in Toulene
Tissues average refractive index 1.53-1.54
Synthetic resins refractive index 1.51-1.55
Best preservative to protect Romanowsky stain when using a natural resin Mineral oil
After cover slipping you notice that the slide has numerous microscopic water droplets all over the slide. How do you fix this? Remove coverslip and any remaining medium with xylene. Rehydrate with the appropriate reagent, clear with xylene, and remount with synthetic resin
Aqueous mounting media is used when Dehydrating and clearing will adversely affect the stain
Aqueous mounting media refractive index 1.41-1.43
Ideal range for photomicrography 1 1/2 (180 micrometers thick), number 1 coverslips (~150 micrometers thick)
As thickness increases Section transparency is reduced
Thickening of the mounting medium does what to transparency? Decreases
Cloudiness of the section Results from thickened medium or excess medium between the section and cover glass
If drying artifact is noted what should be done to correct it? Rehydrate the slides by placing in water for 15-20 minutes. Restain if needed, and dehydrated, cleared and mounted with synthetic resin
Exhibits brown stippling that resembles pigment or nucleus appears ti have a glossy black structure Drying artifact
Created by: Ziek98



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