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

Table 1 Sequential experimental steps for in situ hybridization

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	Protease^a	0.2 mg/ml	Distilled water	15 min	37°C
	RNase A^b	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	RT^d
Denaturation of hybridization solution				4 min	95°C
Hybridization				o/n^c	37°C
Detection of hybrids	Anti-biotin 10 nm gold conjugate	1:25	PBS	30 min	RT^d

^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

ISSN: 1742-4690