CONVENTIONAL TWO STEP FIXATION [See D.H. Hall, 1995]

"CONVENTIONAL" TWO STEP FIXATION [See D.H. Hall, 1995]

1. Wash animals in M9 and anaesthetized in 8% alcohol in M9, 5’ 2. 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M sucrose, 0.05M cacodylate on ice, 2 hours 3. 3 rinses in 0.2M cacodylate on ice, 10 min. each 4. 0.5% OsO4, 0.5% KFe(CN)6 in 0.1M cacodylate, 90 min. on ice 5. 3 buffer washes, 10 min. each, 0.1 cacodylate; 3 buffer washes, 10 min. each (0.1M NaAcetate) 6. Stain in 1% UAc in 0.1M NaAcetate, pH 5.2, r.t., 60 min 7. 3 rinses buffer (0.1 NaAcetate) 5 min. each 8. 3 rinses in distilled water, leave overnight, embed cut pieces in 3% seaplafue agarose 9. Cut out small agarose blocks containing worms; should be large enough to be excluded from pipette tip, but small enough to fit into embedding mold 10. Dehydrate through ethanols; 5 min in 30% ETOH, in 50% ETOH, in 75% ETOH, in 95% ETOH, 3 X 10min. in 100%, 3 X 10 min. ethanol, 3 X 10 min in 100% propylene oxide; all at room temp First change is in 1 part resin to 2 parts propylene oxide, allow to sit 3 hours Second change is in 2 parts resin to 1 part propylene oxide, for 5 hours Then change into fresh resin at least 3-4 times, 2-4 hours per change 11. Place samples in fresh resin at room temp; place samples in air tight containers and agitate or rotate samples slowly. Rotation or agitation of the samples helps with the infiltration. 12. Place samples in fresh resin in embedding mold, and position carefully. 13. Cure samples in 60oC oven for 3 days Resin formula [use pre-dessicated plastic beaker with gradations for easy measurements]: 45 ml Scipoxy 812 Other brands include Embed 812, Medcast, Epon 30 ml DDSA 25 ml NMA Mix components well with wooden applicator add accelerator, 1.5 ml DMP-30 Mix resin thoroughly again Place resin into dessicator and put under vacuum for 5-15 min to remove gasses from mixture before using This protocol includes some minor changes from the method shown in the book chapter: Osmolarity of the aldehyde fix is adjusted with some added sucrose to increase it Potassium ferrocyanide is added to the osmium fix to improve membrane contrast. Osmium tetroxide can be used at 0.5% - 2.0%. Same results can be obtained while fixing at each step at room temperature Exact fixationand staining times are flexible. Since Medcast resin is no longer available, we are using either Scipoxy resin or Embed 812 resin.v