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Table 1 Sequential experimental steps for in situ hybridization

From: Wild-type and central DNA flap defective HIV-1 lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels

Step Material Concentration Buffer Duration Temperature
Fixation Paraformaldehyde 4% Sörensen 1 hr 4°C
Lowicryl embedding     5 days -30°C
Sectioning (gold grids)      
Enzymatic digestion Proteasea 0.2 mg/ml Distilled water 15 min 37°C
  RNase Ab 1 mg/ml Tris HCl, 10 mM, pH 7.3 1 hr 37°C
Denaturation of grid target DNA NaOH 0.5 N Distilled water 4 min RTd
Denaturation of hybridization solution     4 min 95°C
Hybridization     o/nc 37°C
Detection of hybrids Anti-biotin 10 nm gold conjugate 1:25 PBS 30 min RTd
  1. a The aim of this step is to eliminate proteins within the section which could otherwise interfere with binding of the probe to the target DNA. b The aim of this step is to eliminate all RNA molecules including viral RNA to prevent their concomittant detection with viral DNA. c Overnight. d Room temperature