Procedure Chromogenic in situ hybridization

procedure performing chromogenic in situ hybridization


probe design

probe design cish similar fish differences in labelling , detection. fish probes labelled variety of different fluorescent tags , can detected under fluorescence microscope, whereas cish probes labelled biotin or digoxigenin , can detected using bright-field microscope after other treatment steps have been applied.

cish probes approximately 20 nucleotides in length , designed dna targets. complementary targeted sequence , bind after denaturation , hybridization step. few cish probes available commercially, applications have extracted, amplified, sequenced, labelled , mapped bacterial artificial chromosomes (bacs). bacs developed during human genome project necessary isolate , amplify short fragments of human dna sequencing purposes. nowadays, bacs can selected , positioned on human genome using public databases such ucsc genome browser. ensures optimal complementarity , sequence specificity. dna extracted bac clones , amplified using polymerase-based technique, such degenerate oligonucleotide primed (dop)-pcr. next, clones sequenced , position on genome verified. probe labelling can carried out using either random priming or nick translation incorporate biotin or digoxigenin.

preparation of tissue, hybridization of probes, , detection

for cish work optimally, chromosomes must in either interphase or metaphase. tissue samples securely attached surface, glass slide, paraffin. tissue samples must washed , heated several times remove paraffin before hybridization step. after this, sample has undergo pepsin digestion ensure target accessible. final step, 10–20 μl of probe added, sample covered coverslip sealed rubber cement, , slide heated 97 °c 5–10 minutes denature dna. slide placed in 37 °c oven overnight probe can hybridize. on next day, sample washed , blocker nonspecific protein binding sites applied. if horseradish peroxidase (hrp) going used, sample must incubated in hydrogen peroxide suppress endogenous peroxidase activity. if digoxigenin used probe label, anti-digoxigenin fluorescein primary antibody followed hrp-conjugated anti-fluorescein secondary antibody applied. if biotin used probe label, non-specific binding sites must first blocked using bovine serum albumin (bsa). then, hrp-conjugated streptavidin used detection. hrp converts diaminobenzidine (dab) insoluble brown product, can detected in bright-field microscope under 40- 60-fold magnification. counterstain such hematoxylin , eosin can used make product more visible.