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T. Culture cells mitotic apparatus

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

Last Updated: 10/2/08