4.8. Histochemical analysis

Gene transfer mediated by the AdCMVlacZ virus was monitored by histochemical analysis of lacZ gene expression on cryosections. Ten pieces of tissue were taken randomly from the perfused organ (kidney, spleen and lung) and snap-frozen in liquid nitrogen. From every piece, several sections were prepared for analysis. Ten to 32 sections per organ tissue per experiment were examined. Cryostat sections 5 µm thick were first fixed for 10 minutes in 4% glutaraldehyde. Following extensive washings with 1 x PBS, the sections were incubated in a detergent solution containing 0.01% sodium deoxycholate, 0.02% NP40 and 2 mM magnesium chloride in PBS for 10 minutes. The sections were incubated in an X‐gal solution (detergent solution containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg ml^–1 X‐gal (5‐bromo‐ 4‐chloro‐3‐indolyl β -d-galactopyranoside)) for 3 hours or overnight at 37°C and subsequently counterstained with PAS. In the kidney and lung, the location of expression was additionally determined by immunohistochemical staining. Possible cytotoxic and inflammatory changes were evaluated by histologic examination of formaldehyde‐fixed and paraffin-embedded tissue sections after HE, PAS, Perls` iron, May-Grünwald-Giemsa, van Gieson`s and Verhoeff` elastic stains.

The distribution of transgene expression was evaluated by examining visually the nuclear-dominant blue color areas of X-gal staining in the different cell types of the kidney, spleen and lung. In the kidney, the proportion of transgene-expressing glomeruli out of the total number of glomeruli per section was calculated. In lung tissue, the quantification of expression was estimated by counting all bluish spots showing transgene expression in all of the 20 sections (area 100 mm² per section).