Cryosectioning technique

Slide-Pre-Treatment

• 1. Place slides in slide rack

  2. Place into chromic-sulphuric acid (glass cleaner) for 2 hours

  3. Place under running tap water until water is completely clear

  4. Let slides dry

Gelatin Coating

• 1. Add 5g of gelatin to 500 ml of heated dH2O (60^oC)

  2. Add 0.425g of chromium alume and stir

  3. Let cool down slowly to room temperature

  4. Dip slides slowly 3X

  5. Let dry in 37^oC incubator overnight

Cryosectioning Scaffolds

1. Rinse scaffolds in ddH2O (3X 15 min) and place into wells of a 12 well culture plate.

2. Completely cover scaffolds with tissue freezing media (TFM) (Fisher Scientific) and place under a vacuum at 635 mm of Hg for 4h

3. Cryosectioned at –30oC (Ames Lab-Tek cryostat, Elkhart, IN).

a. I found 30 mm sections worked best

4. Maintain sections at 40 oC overnight on a slide warmer.

5. Prior to staining, immerse slides in water for 10 minutes to remove the TFM.

a. you may need to play around with this step as it is necessary to remove all TFM but you also do not want your sections to detach from your slides.

b. I found that dipping individual slides into and out of the water after being submersed for 10-15 min in the water worked best. This way you can keep an eye on your sections to determine how much punishment they can handle J.

6. Stain sections with either Gomori’s trichrome or toluidine blue.