Molecular Techniques and Methods

Paraffin Infiltration and Sectioning of Plant Tissue

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

After fixing, the water must be removed from the tissue before it is embedded in a water-insoluble matrix such as paraffin. Dehydration is accomplished by exposing the tissue to higher concentrations of ethanol or acetone. Tertiary Butyl Alcohol (TBA) is routinely used for paraffin material because paraffin is miscible with TBA.




MATERIALS AND SOLUTIONS




PROCEDURES

1. From the 50% ethanol, dehydrate the tissue according to the TBA dehydration series as follow.

Step
100% Ethanol
TBA
Distilled H2O
Time
A
50 ml
10 ml
40 ml
30-60 min
B
50 ml
20 ml
30 ml
30-60 min
C
50 ml
35 ml
15 ml
30-60 min
D
40 ml
55 ml
5 ml
30-60 min
E
25 ml
75 ml
------
30-60 min
F
------
100 ml
------
Overnight

  • TBA solidifies at room temperature (25.5oC), so place the vials with absolute TBA on top of an oven or in another warm place.

    2. Follow with three changes of absolute TBA in Step F, keep the tissue in one change of absolute TBA overnight.

    3. Material should now be transferred to aluminum weighing dishes and filled 1:1 (v/v) with 100% TBA and molten paraffin.
  • The paraffin should be filtered to make certain that it is particle-free.

    4. Place the aluminum weighing dishes in a warm oven (60oC) overnight.
  • As the TBA evaporates, the paraffin will gradually infiltrate the tissue.

    5. Pour off any remaining mixture, and add fresh melted paraffin.

    6. Make one change with additional paraffin, and let the paraffin infiltrate the tissue overnight.

    7. Make 2-3 more changes of molten paraffin (every 4 hours). The material is now ready to embed.

    8. Have tools warm (use an alcohol lamp).
  • Quickly transfer the aluminum dishes to a warming table and orient the tissue with warmed needles.
  • When oriented, gradually move the dishes to a cooler area of the warming table.
  • Allow the paraffin to solidify and cool completely at room temperature before transfering the embedded tissue to 4oC.

    9. Store at 4oC until use.

    10. Once a trimmed specimen and knife have been mounted in the microtome, sectioning can begin. Position the specimen before putting the knife into place.

    11. The block face of the specimen can either have a rectangular or a trapezoidal form, but in either case, make sure that the block is exactly parallel to the knife edge. Thus, as each section is cut, it will press evenly upon the trailing edge of the previous section and detach it cleanly from the knife edge with a minimum of wrinkling. Check that there is no grease, dust, or other contaminants on the block face or knife edge, as these may damage the tissue.

    12. Move the wheel of the microtome with a steady, even stroke.
  • Hold the camel's hair brush in one hand to lift ribbons of sections as they come off the knife.

    13. Spread the ribbons in an appropriate container, or place them directly onto the slides.

    14. Examine the sections under the dissecting microscope to determine which sections you wish to affix to a slide.

    15. Prior to affixing them to the slides, sections can be stored overnight or longer in the storage box at 4oC.

    16. To affix the sections to the slides, place several drops of DEPC-treated H2O on the slide.
  • Use positively charged slides, which are commercially available.

    17. With a brush, place the sections on the flooded area, and very gently pull on their edges with dissecting needles to align them on the slide.

    18. Place the slide on a warming table (40-50oC) until any wrinkles in the ribbon are removed.

    19. Remove excess liquid with Kimwipes, and dry the slides overnight on the warming table in a dust-free area.

    20. Transfer the slides to a slide box and keep them at 4oC until use.
  • Slides can also be deparaffinized at this point by putting them in a slide rack and subjecting them to two changes of xylene (in a fume hood), 30 min each.

    21. Remove the slide, tray to paper towels.
  • After the xylene has evaporated, the sections may be examined with the microscope to determine if they are suitable for hybridization.
  • The slides can be stored at 4oC indefinitely.




    NOTES

    Overfixation
  • Poor hybridization may be caused by inaccessibility of tissue mRNA. To prevent this, decrease the fixation time.

    Underfixation
  • Can lead to diffusion of tissue mRNA as well as poor tissue preservation. The optimal time must be determined empirically for each tissue type. Loss of tissue sections during the hybridization steps may be due to poor quality slides. Use positively (+) charged slides, which are commercially available.




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    REFERENCES



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