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This method specifically highlights centromeric regions of all chromosomes and produces distinct staining pattern on horse sex chromosomes (Figure 22.4). C‐banding is used to further verify the number and morphology of sex chromosomes. The method was originally developed by Arrighi and Hsu (1971) and includes the following steps:

 Treat the slides with 0.1 N HCl at room temperature for 3 minutes and rinse in distilled water.

 Treat the slides in 5% Ba(OH)2 at 60°C (140°F) for 3 minutes and rinse in distilled water.Figure 22.4 A C‐banded metaphase spread of a male horse; distinct C‐bands of the X and the Y chromosomes are highlighted at the lower right.

 Treat the slides for 5 minutes in 2× SSC at 60°C and rinse in distilled water.

 Stain the slides in 4% Giemsa in Gurr buffer for 1 hour, rinse gently in distilled water, and air‐dry.

Note: timings of treatment steps for C‐banding can vary between slides and individuals and may need adjustment.