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EM

Lab/Staining

QuestionAnswer
Well-fixed plasmalemma: Is complete w/ no breaks in membrane
Well-fixed nuclear envelope: Is uniform, undilated space between inner & outer membranes
Well-fixed mitochondria: Have no swelling, disruption, v. sensitive to quality of fixation
Well-fixed endoplasmic reticulum: Has regular width & arrangement of cisterns/channels
Well-fixed cytoplasm: Is finely precipitated, not too obvious in most cells, well-preserved appears more complex than poorly preserved
Well-fixed nucleus: Varies w/ type of fixative from finely granular w/ osmium to aggregated chromatin masses w/ aldehydes
2 categories of fixatives used: OsO4 & aldehydes; Zamboni PAF not widely used but is good for light & EM, if used usu. post-osmicate specimens
Advantages of primary OsO4 fixation: Excellent preservation cytological detail; renders lipids insol. - good membrane preservation
Disadvantages of primary OsO4 fixation: Specimens in fixative max 2-4 hour; poor penetration, specimens must be about 1 mm cubes; can't perform histochemical study
Advantages of primary aldehyde fixation: Better penetration, easy perfusion; can perform histochemical study; specimens still okay after in formaldehyde for long time; formaldehyde & some formaldehyde-glutaraldehydes dual-purpose; best cellular preservation when post-osmicated
Disadvantages of primary aldehyde fixation: Lipids not preserved unless post-osmicated; membrane-bound cavities tend to be enlarged; membranes are e- lucent unless post-osmicated
Advantages of primary buffered PAF fixation: Specimens can remain in fixative indefinitely; rapid penetration, stabilizes proteins dual-purpose
Disadvantages of primary buffered PAF fixation: Lipids not well-preserved unless post-osmicated; some cytoplasmic granules & lysosomes not preserved; some background substances not well-preserved
Factors influencing fixation: pH - 7.2 to 7.4, temp. - traditionally 4°C, b/c swelling, now room temp., tonicity - 300 mOsm, dextrose & sucrose to adjust, length of fixation - formaldehyde, PAF, formadehyde-glutaraldehyde - indefinitely, glutaraldehyde - 2-4 hours, OsO4 1-2 hours
Commonly used pH buffers: Phosphate, cacodylate, s-collidine, veronal acetate
Paraformaldehyde w/ cacodylate buffer fixative reagents sodium cacodylate dH2O HCl paraformaldehyde NaOH CaCl pH 7.3 to 7.4
Paraformaldehyde or glutaraldehyde w/ phosphate buffer fixative reagents monobasic sodium phosphate NaOH paraformaldehyde or glutaraldehyde pH 7.2 to 7.4
Formaldehyde w/ phosphate buffer (Modified Millonig Fixative) fixative reagents formaldehyde dH2O sodium phosphate monobasic NaOH pH 7.2 to 7.4
Formaldehyde-glutaraldehyde fixative reagents monobasic sodium phosphate NaOH formaldehyde glutaraldehyde
Buffered PAF (Zamboni) fixative reagents paraformaldehye picric acid NaOH phosphate buffer dH2O pH 7.3, 900 mOsm
OsO4 w/ cacodylate buffer fixative reagents OsO4 dH2O sodium cacodylate w/ dH2O & HCl (buffer) CaCl2 pH 7.2 to 7.4, store @ 4°C
OsO4 w/ phosphate buffer fixative reagents OsO4 sodium phosphate, monobasic NaOH dH2O dextrose pH 7.3 to 7.4, store @ 4°C
EM processing Similar to light processing, embedding media not miscible w/ water so must dehydrate & carry thru transitional solvent (similar to clearing agent)
EM dehydration Usually ethanol, can use acetone, dioxane, 2-ethoxyethanol, dimethyl formamide
Transitional solvents Propylene oxide w/ epoxy resins, (can use w/ polyester resins), styrene best w/ polyester resins
Embedding media Methacrylate embedding - old, enhances specimen contrast so no stain needed, lose some cellular detail; Vestopal W - polyester resin, sections easily, stains well, difficult to obtain; Epon, Araldite, Spurr epoxy resins most common
Routine processing & Spurr embedding 0.5 cm thick tissue in 20x vol. of fixative, prefer overnight, core biopsy of tissue, remove damage, OsO4, 50% ethanol x2, 70%, 95%, 100% ethanol, decant, add epoxy, polymerize 12 hours, 60°C
ERL 4206 Vinyl cyclohexane resin, poxy resin
DER 736 Diglycidyl ether of propylene glycol, flexibilizer for epoxy resin
NSA Nonenyl succinic anhydride, ardener for epoxy resin
DMAE Dimethylaminoethanol, accelerator/catalyst for epoxy resin
Routine processing & Epon embedding 0.5 cm thick tissue in 20x vol. of fixative, prefer overnight, core biopsy of tissue, remove damage, OsO4, 50% ethanol x2, 70%, 95%, 100% ethanol x2, propylene oxide, 50:50 propylene oxide-catalyzed resin, catalyzed resin, polymerize 12 hours 60°C
DDSA & MNA Dodecenyl succinic anhydride, methyl nadic anhydride, hardeners for Epon resin
DMP30 Tridimethylaminomethyl phenol, accelerator/catalyst for Epon resin
LR White Hydrophilic acrylic monomer, must fix specimens w/ aldehyde as soon as blood cut off so well-preserved antigenic sites
Processing w/ LR White for EM Immunolabeling 1 mm tissue, fix 1-4 hours, max 12, wash w/ cacodylate or phosphate buffer, 60% ethanol x2, 80% ethanol x2, 95% ethanol x2, 2:1 LR White:ethanol fresh resin x3, embed w/ BEEM or gelatin capsules w/ fresh resin, polymerize 50-55°C 20-24 hours
BEEM Better Equipment for Electron Microscopy
For immunolabeling resin must be cured: Thermally, 50-55°C 20-24 hours, not accelerator
When are specimens are not post-fixed with OsO4? When preparing for immunolabeling, OsO4 has harsh, deleterious effect on antigenicity
Sectioning tips: No vibrations, no drafts; trim block so smallest face possible; trapezoid face; lens paper to clean trough of debris; finger oil can contaminate tools & sections; use oil-free blades or remove oil w/ acetone & water
Section thickness 50-90 nm, thick sections appear purple, blue, green, yellow, gold sections are about 90 nm thick, silver sections are about 50 nm thick, dull gray sections too thin
Diamond knives Used for most thin sections, handle carefully, clean after each use, good to have 1 per person, use different knives for different specimens
Glass knives Cut 0.5 µm sections, must break shortly before use
"Histo" knives Low-grade diamond knives, 5-mm cutting edge, w/ proper care edge lasts long w/o sharpening
Average clearance angle 2° to 5°
Care of diamond knives Don't touch edge, avoid solvents in trough, don't let section dry on edge - if do soak w/ dilute nonionic detergent of neutral pH immediately remove unused sections, clean only w/ tools for diamond knife so don't chip edge, don't use sonication
Problem - sections of varying thicknesses: Check tightness of block, knife holder, knife, change to different area of knife or resharpen, cut faster or slower, soft block - heat @ 60°C for 24 hours, drafts, vibration, keep steady rhythm
Problem - skipped or uncut sections: Reset microtome advance, change to different area of knife or resharpen, tighten knife & block, wet block - dry w/ lens paper, soft block heat @ 60°C for 24 hours, vibration, keep steady rhythm
Problem - chatter/undulations in sections: Reduce speed, reduce clearance angle, reduce size of block face, vibrations
Problem - sections crumble, stick to knife edge: Raise meniscus level of trough fluid, clean knife edge (glass knife - discard), increase clearance angle, clean block face w/ lens paper & alcohol
Problem - section lifted by block: Lower meniscus level of trough fluid, dry block face w/ lens paper, increase clearance angle, clean knife edge, block face may be electrified - increase humidity or wet face, check back of knife for fluid droplet, dry
Problem - split sections or lengthwise lines in sections: Nick in knife cutting edge, clean knife edge, block contains glass or dirt - discard block or use old knife
Problem - ribbon is curved: Upper & lower block edges not parallel - retrim, block sides not parallel - retrim
Problem - face of block gets wet: Lower meniscus level of trough fluid, dry back of knife cutting facet, clean knife edge & block face, dry block face w/ filter paper, increase room humidity
2 types staining: Thick, 0.5 µm, sections for light microscope - true staining, thin sections for EM - heavy metal "stain" to enhance e- contrast
Toluidine Blue-Basic Fuchsin staining procedure Thick, 0.5 to 2.0 µm sections, true green-colored section ideal, section on drop of dH2O on glass slide, evaporate water on hot plate, add several drops staining solution, pinch of sodium borate, wash excess w/ dH2O, blot dry, air dry
Toluidine Blue-Basic Fuchsin staining solutions toluidine blue basic fuchsin 30% ethanol
Toluidine Blue-Basic Fuchsin staining results nuclei - dark purple cytoplasm - pink to lavendar fat - gray-green to gray-blue rbc - magenta
Thick sections should be coverslipped w/: Low viscosity toluene-based acrylic resin
Toluidine Bue staining procedure Thick, 0.5 to 2.0 µm, sections, true green-colored section ideal, section on drop of dH2O on glass slide, evaporate on hot plate, stain equal parts 2% T blue, 2% sodium borate on 65-95°C hot plate 1-2 min, rinse w/ dH2O, dip abs. alcohol, air dry
Toluidine Blue staining results nuclei - dark purple cytoplasm - lavender fat - gray-green to gray-blue rbc - deep blue to purple
Thin section stain solution boiled dH2O lead nitrate sodium citrate NaOH good for several months
Thin section stain procedure Section on 200-mesh copper grid, air dry, stain w/ uranyl acetate w/ 50% ethanol 1-3 min., dip 50% methanol, 6+ changes dH2O, dry w/ filter paper, stain w/ lead citrate 1-3 min in Petri dish section-down, 6+ changes dH2O, dry w/ filter paper
To prepare Petri dish for thin section staining: Pour thin layer molten paraffin into clean Petri dish, allow to cool, add 1-2 dozen NaOH pellets, cover & let stand, will absorb CO2
When staining thins w/ lead citrate important to avoid: CO2, causes stain to precipitate as PbCO3 on grids, this includes atmospheric CO2 & breath
Blood cell preparation part 1 For ultrastructural studies on platelets, leukocytes, or rbc: mix venous blood w/ EDTA or heparin by gentle inversion, centrifuge 1000 RPM 10 min, pipette out supernatant & layer phosphate-buffered glutaraldehyde over buffy coat - fix 30 min.
Blood cell preparation part 2 disk now layered w/ desired cells, slice disk thinly, place in phosphate-buffered osmium fixative, fix & process by routine method
Cell suspensions (fluids, cultures, parasites) part 1 Centrifuge well-fixed suspension 1000 RPM 10 min, decant supernatant, resuspend cells in 1mL fixative or buffer solution, w/draw suspension w/ tuberculin syringe, inject cells into bag formed by heat-sealing Nuclepore filter
Cell suspensions (fluids, cultures, parasites) part 2 For TEM routinely process bag as though tissue, flat embed specimens, after polymerization use scope to ID cell clusters, cut out & glue to block, for SEM may critical point dry bag, open, & mount cell side up
Processing tissues previously embedded in paraffin If original fixative good for EM, w/ sharp blade remove desired tissue from block, xylene x2 to remove paraffin, rehydrate: ethanol x2, 95% ethanol, 70% ethanol, 50% ethanol, phosphate buffered formaldehyde, fix w/ OsO4, process routinely
Processing tissue from H&E stained paraffin section part 1 Remove coverslip & mounting medium, rehydrate thru alcohol to water, phosphate-buffered formaldehyde overnight, cut slide to size, flood w/ OsO4 30 min, process routinely, embed section side up in plastic container w/ layer of fresh catalyzed resin
Processing tissue from H&E stained paraffin section part 2 Polymerize 60°C 18 hours, separate slide by immersing in liquid nitrogen, cut portion of tissue desired, glue to epoxy block w/ epoxy glue, may stain entire section on warming plate w/ T-blue-basic fuchsin, section carefullly - thin tissue
Created by: CCF
 

 



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