Box: Slide pretreatment with pepsin to remove cytoplasm

Taken from: Spectral karyotyping analysis of human and mouse chromosomes

Published in: Nature Protocols

"Check the slide under a light microscope using a times40 high-dry phase objective. If cytoplasm is present (gray particulate matter surrounding the chromosomes; see Fig. 2a), pretreat the slides with pepsin as described in Box 1. If cytoplasm is not visible (a clear light margin surrounds the metaphase chromosomes) and the chromosomes have good morphology (black in color, not phase-light or shiny), there is no need for slide pretreatment with pepsin." (Quoted from the protocol)



Effects of humidity and removal of excess cytoplasm by slide pretreatment on hybridization efficiency.

(a) When cells are not kept in the hypotonic solution for enough time, the chromosomes become trapped inside the cytoplasm (white arrows). Altering the humidity at which the metaphase preparation is dropped onto the microscope slide may sometimes alleviate this problem; otherwise, hybridization efficiency will be compromised. (b) When normal human metaphase spreads are pretreated with pepsin and hybridized with a whole chromosome paint probe labeled with tetramethyl rhodamine isothyocyanate (TRITC), the hybridization will be successful. (c) Here, a whole chromosome paint-labeled with TRITC was hybridized to a different normal human metaphase spread and the slide was not pretreated with pepsin. Note the weaker intensity of the hybridization signal (red color).