| 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 |
