Identification and phenotyping of infected cells from female reproductive tract. a,

<p>48 hours after inoculation with JRFL pseudotyped LICh vector, the reproductive tract is separated into large sections (panel 1; V1: labia and lower vagina; V2: upper vagina; C: cervix; U: uterus; O: ovary), soaked in d-luciferin to identify luciferase expressing regions with in vivo imaging systems, and dissected into sequentially smaller pieces (panels 2–4) to isolate strongly luminescent regions measuring 2×2 mm². (animal code: EH99) <b>b,</b> Left panel; The spectral emission profile of a putative infected cell from the tissue identified in <b>a</b> is measured on laser scanning confocal microscope with resonant scanner. Emission profile matches the defined profile of mCherry. Right panel; Pseudo-colored reconstructed image on the right shows the localization of mCherry (red), DAPI (blue) and background signals (green). <b>c,</b> Vaginal tissue is stained for firefly luciferase expression. The combination of mCherry⁺ (red), TRITC^−/low (red), luciferase⁺ (red) fluorescence is criteria for confirming infection. The right panel shows relative fluorescence intensity of mCherry, TRITC, luciferase, and nucleus is along the line in mCherry image. (animal code: HP79) <b>d,</b> Phenotyping of infected cells by surface marker staining, shown in green, for CD3 (T Cell), CD4 (HIV-1 primary receptor), or CD68 (tissue resident macrophages) shows all conventionally defined HIV susceptible cell types are transduced within the vaginal tissue shown in <b>a</b>. (animal code: EH99).