Improved in situ hybridization and G-banding by pretreatment with Denhardt's solution and gelatin-chrome alum.
Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity during in situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline-citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 X SSC for 2 min at 70 degrees C. A 3H-labelled, cloned DNA fragment of the highly repetitive AGM component alpha DNA was hybridized to the chromosomes by incubation at 45 degrees C for 16 h. Treatment with gelatin-chrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 X Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin after in situ hybridization.
Lee
G M
GM
Rasch
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EM
Musich
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PR
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002857:Chromium; D017608:Chromium Compounds; D002871:Chromosome Banding / Q000379:methods*; D002875:Chromosomes / Q000032:analysis*; D005780:Gelatin; D008677:Metaphase; D009693:Nucleic Acid Hybridization; D017680:Potassium Compounds; D011489:Protein Denaturation; D013194:Staining and Labeling; D013431:Sulfates
D017608:Chromium Compounds; D017680:Potassium Compounds; D013431:Sulfates; D002857:Chromium; C020720:chrome alum; D005780:Gelatin