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TEM/IEM Flat Embedding and Serial Section
Protocol for Duane Compton's lab, Biochemistry Dept.
All fixation steps are done at room temperature (RT).
1. Tissue culture cells on coverslips (25mm etched glass from Bellco Glass),
extracted to expose the mitotic(this part done in Duane's lab) are fixed in 2%
GTA in 0.1M Na Cacodylate buffer pH 7.4 @ 1 hr. at RT ( or overnight, if
necessary).
2. Rinse three times in 0.1M Na Cacodylate buffer pH 7.4. Transfer to new
petri dishes.
3. Post-fix in 1% OsO4 in 0.1 M Na Cacodylate at pH 7.4 for 30 mins. - 1 hr.
at RT.
4. Rinse in 0.1 M Na Cacodylate at PH 7.4. Transfer to new petri dishes.
5. Rinse 2 times in dH2O.
6. ****En-Bloc stain in 2%UA aq for 30 mins., in the dark at
RT.
****Don't Skip. Important for Ultrastructural
preservation
7. Rinse in dH2O.
8. Dehydrate through ETOH series: 30%, 50%, 70%, 85%, 95%, 3-5 mins. ea.
9. Three 5 min. changes in 100% ETOH.
10. Two 5 min. changed in Propylene oxide (PO) in 60 mm. glass petri-dishes.
Make sure coverSlips stay covered with fluids at all times.
Do 4x changes, if necessary.
11. Two changes of Epon-Araldite:PO - 1:1 mixture. Make sure coverslips
stay covered with fluids at all times. Do 4x changes, if
necessary. Coverslips are QUICKLY transferred to
flat-embedding slide mold and covered with Epon-Araldite:PO - 1:1 mixture.
12. The flat-embedded samples are left overnight, in a desiccator (NO
Vacuum), to remove PO.
13. Fresh resin from the same batch is removed from freezer and thawed for @
1 hour; Coverslips are carefully removed from flat-embedding slide mold. Excess
epon/araldite is removed from bottom surfaces, by wiping with cotton swabs
(make sure to keep track of which side is CELL SIDE UP!!. Coverslips are placed
in clean flat-embedding slide mold and a thin layer of fresh epon/araldite is
added, by drops to the surface of the coverslip. Leave for one hour.
14. Coverslips are placed in a 60ºC oven and fully polymerized, 24 hr.:
total time for Epon/Araldite and 48 hr. total time for pure Epon.
15. Remove coverslips from heat. Check bottom surface of coverslip. GEMTLY
remove any epoxy layer from the bottom surface with fine sandpaper(120-2400
grit) This will expose the glass surface. Glass is removed by etching in cold
(store HF in fridge 24 hrs. before use) concentrated hydrofluoric acid. Once
the glass is etched away, the plastic wafer is rinsed thoroughly with distilled
water and then dried at 40ºC for one hr. Etching times: Bellco etched glass
coverslips - 2.5 to 4 minutes, depending on temperature of HF (4ºC-20ºC). Be
careful not to scratch surface during water rinse. Works 100%, with a nice,
clean surface.
16. Mark area of interest, using a dissecting scope. Place epoxy square
inside slotted petri-dish; use single edge razor; tap GENTLY with hammer;
slotted petri-dish should keep cut pieces from "flying" across the lab bench.
Attach pieces to an epoxy blank with crazy glue. Mark each block with the two
letter code, for reference.Check block for strong attachment, then CARFEULLY
trim away excess epon around chosen cell or cells. In this case, each etched
square is @ 1 mm2 and the entire perimeter should be preserved:
Bellco Glass co.
PO Box B
340 Edruduo Road.
Vineland, N.J. 08360
1-800-257-7043
Each etched square is @1mm with 2 letters or 1 letter/1 number marking
center area of each square.
EPON/ARALDITE:
10ml. LX112(POLYBED812, etc.)
10ml. Araldite 502
24ml. DDSA
0.9ml DMP 30 (added just before use)
Mix together first three by shaking vigorously. Warm in 50ºC oven for 5-10
minutes, to improve mixing. Add DMP-30 and mix vigorously. Resin mix may turn
orange, depending on pH of DMP-30. Remove air bubbles with vacuum for @ 30
minutes. Store aliquots of resin mix in 5 or 10ml disposable syringes in -20ºC
freezer; will keep for 1 week. Be sure to use same resin mix for all steps of
infiltration.
Section:
Cut 0.12-0.15(purple) SERIAL micron sections (no thicker). Collect sections
in order. record each section number and description. Record any missing
sections. Collect sections on 400HH copper grids for maximum open area, without
having to use slot grids.
Stain(using Hiraokastaining kit[cat#4635, PolySciences, Inc.] : Ua aq for
45' at 50ºC and Reynold's LC 20' at RT.
For IEM sections: Stain(using Hiraokastaining kit[cat#4635, PolySciences,
Inc.] : Ua aq for 45' at 50ºC NO LC staining
References:
Dionne, M., L. Howard, D. Compton, 1999, NUMA is a Component of an Insoluble
matrix at the Mitotic Spindle Pole, Cell Motility and Cytoskeleton
42:189-203.
Howard,L., D. Compton , A.Saredi, 1997, Transmission electron microscopy
flat embedding technique, Microscopy Today 97(7): 24-25
Moore, M.J., 1975, Removal of glass coverslips from cultures flat embedded
in epoxy resins Using HF, . Microscopy 104: 205.
Mountain, V., C. Simerly, L. Howard, A. Ando, G. Schetten, D. Compton 1999,
"The Kinesin-related Protein, HSET, opposes the activity of Eg5 and cross-links
microtubules, J. of Cell Biology 147(2):351-365.
Rieder, Conly and Bowser, Samuel, 1987, "Correlative LM and EM on the same
epoxy section", in Correlative Microscopy in Biology , Chapter 11,
Academic Press, 1987.
Saredi, A, L. Howard, D. Compton 1996, NUMA Assembles into an extensive
filamentous structure when expressed in the cell cytoplasm", J. Cell
Science 109:619-630
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