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EM
Lab/Staining
| Question | Answer |
|---|---|
| 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 |
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CCF
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