Immunofluorescence detection of parvalbumin in the heart. (a–h) The photographic montage assembled from representative serial cryosections extending from the apex to the base of a single heart at day 6 after gene transfer (representative of four experiments). (a and b) LV base sections; (c and d) LV midsection; (e–g) LV apical sections; (h) RV section. (h, inset) LV apical section from normal saline-injected control heart. Photographic exposure time was the same in a–h. There was extensive, parvalbumin-immunopositive reactivity in myocytes spanning the width, depth, and length of the left heart (apex to base). The primary antibody was anti-parvalbumin (PARV19; titer 1:500–1:1,000; Sigma Chemical Co.) and the secondary was goat anti-mouse IgG conjugated to Texas Red (titer 1:100; Molecular Probes Inc., Eugene, Oregon, USA), using methods described elsewhere (27). Dual labeling of single cells isolated from these hearts showed the colocalization of parvalbumin and sarcomeric actin, indicating that the cardiac myocytes were being transduced. Scale bar = 100 μm, a–h. (i) Low-power photomicrographs documenting the extent of lacZ gene expression (using X-gal, which results in blue staining of positive cells) after direct gene transfer to the heart in vivo. All panels are taken from the same gross dissection (sectioned in thirds) of a heart. Clockwise from left panel: side-view of apical one-third of heart with LV in foreground, demonstrating extensive transmural staining; side view of middle one-third of heart; side view of reconstruction of the apex/mid/base sections together; top view of midsection with base-facing section up (in this view, the spread of intense B-gal staining is evident across LV wall, with little or no detection in IVS or RV).