Begin by excising at least 20 testes from the pupae of Anopheles mosquitoes in sterile PBS. Transfer the testes to a clean microscope slide containing a fresh drop of PBS. Using P1000 boarded filtered tips or the tip of a dissection needle, transfer the testes into an embryo dish containing 3.7%formaldehyde in PBST.
Incubate for 10 minutes at room temperature. Next, wash the testes in PBST for five minutes at room temperature. Transfer the testes to the embryo dish containing RNase A and incubated for a half hour at 37 degrees Celsius.
Remove the RNA solution and add one milliliter of the penetrating solution. Incubate the testes in the penetrating solution at room temperature for 10 minutes. Then wash the testes and PBST two times for five minutes each.
Transfer the testes to a 1.5 milliliter tube containing 20 to 30 microliters of hybridization solution with the labeled DNA probe. Incubate the mixture overnight at 37 degrees Celsius in the dark to allow hybridization. With the help of AP 1000 white bore filtered tip, transfer the testes into an embryo dish, then wash them in two XSSC for five minutes.
Remove the SSC then mount the testes on a frosted glass slide using a commercially available mounting medium containing DAPI. Seal the slide with a cover slip sealant and incubate it at room temperature in the dark for two hours. A good level of hybridization allowed for the visualization of the targeted chromosomes throughout spermatogenesis.
The pairing of the labeled sex chromosomes could be seen in the prebiotic and myotic stages. X or Y bearing spermatids could be followed throughout spermiogenesis marked by different levels of DNA condensation to the final step of arrow shaped mature spermatozoa.
