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Kidney Tubule Cells from Mice

Susan Kennedy--- Peter Friedman's Lab in Physiology

Fixation:

Cells were grown on 30mm collagen filters or 30mm glass coverslip. In future, request use of 13mm cover slips; 30mm cover slips are too large to work with.

All but one of the Collagen filters were immersed in GTA fix, while still attached to their plastic holders. Filters will be removed after secondary fixation and water rinse, by cutting collagen filter away from plastic holder and lifting holder out of dish, leaving the filter behind. Special large nitex envelopes will be used to hold the collagen filters during critical point drying. The glass cover slip will be held in a re-molded metal coverslip slot holder.

1.Primary Fix: 2% GTA in 0.1 M NaCacodylate Buffer, pH 7.6 for 2-3 hrs., with several changes over the fixation time.

2. Buffer rinse: 0.1 M Na Cacodylate Buffer, pH 7.6 three times, 10 minutes each.

3. Secondary Fix: 1% OsO4 in 0.1 M Na Cacodylate Buffer, pH 7.6 for 2 hr at room temperature.

4. Buffer rinse: 0.1 M Na Cacodylate Buffer, pH 7.6, 10 minutes.

5. Dehydrate: 50% ethanol for 10 minutes, 70% ethanol for 10 minutes, 95% ethanol for 10 minutes; 3 changes of 100% ethanol over one hour.

6. CPD.

7. Carbon coat with 100? indirect carbon.

8. Sputter coat with 100? AuPd.

References:

McAteer, J.A. et al (1986), Scanning Electron Microscopy of Kidney Cells in Culture, SEM III pp.1135-1150

McAteer, J.A. et al (1987), Morphogenetic Clonal growth of Kidney Epithelial Cell Line, Anat. Record (217) pp.229-239

McAteer, J.A. et al (1988), Explant Culture of Human Polycystic Kidney, Laboratory Investigation, Vol59 #1 pp.126-136

Last Updated: 10/2/08