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Extra resources for Pancreatic Cancer: Methods and Protocols (Methods in Molecular Medicine)
3. Hemocytometer cover slip or other device narrower than glass slide to use as a cell spreader. 4. 70% ethanol. 3. Tissue Scraping Fresh tissues containing cells that can be detached intact with mild shearing force can be rapidly sampled by scraping with a sterile scalpel blade and then rapidly spreading the scraped liquid sample onto a glass slide with the blade. A basic requirement for use of this technique together with microdissection is that the intended target cells can be readily identified cytologically or by cytology together with specific staining characteristics.
2. Place bed of dry ice in ice bucket. 3. Place the metal container containing isopentane onto dry ice. Allow 15–30 min for a cool-down period. Put the tissue to be frozen into the metal or Plexiglas basket long enough for complete freezing, Excess isopentane (highly flammable) can be removed with a paper towel (see Note 16). 2. Gentle-Jane® Method In our hands, because of the rapidity of freezing, this method provides superior frozen section histology, and in some cases provides as good as or better histology than from formalin-fixed, paraffin-embedded samples (see Note 17).
3. Drop immediately (before drying) into fresh 70% ethanol for 10 dips. Then airdry or proceed to staining. 3. Tissue Scraping (see Notes 22 and 23) 1. Gently scrape a sterile blade against wet tissue surface to collect fluid on the edge of the blade. 2. Drag the blade gently across the dry glass slide. 3. Immediately plunge into 70% ethanol for 20 dips, then proceed to staining or airdry. 4. 1. DENSITY AND SIZE GRADIENT SEPARATIONS (SEE NOTE 24) 1. Dilute cells, and prepare gradient in 50-mL conical tubes as directed by the manufacturer.