Immunofluorescence staining of sarcoidosis AMs showing presence of HIF-1α in the cytoplasm and nuclei. AMs (1 × 105) were allowed to adhere on chamber slides overnight. The cells were washed with PBST and fixed with 3.7% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100, blocked (10% FCS), and then incubated with anti-HIF-1α antibody overnight at 4°C. The secondary antibody was Alexa-fluor 488 - conjugated goat anti-rabbit antibody. Images were analyzed by immunofluorescent microscopy (AX10, Zeiss). Images show nuclei staining of AMs (A), overlay image shows nuclear and cytoplasmic co-localization of HIF-1α (Β). Confocal laser scanning microscopy (CLSM-310, Zeiss) images show nuclei stained with DAPI (blue) in a single AM (C) and a multinucleated giant cell (D), nuclear and cytoplasmic accumulation of HIF-1α in green (E and F), overlay image shows nuclear co-localization of HIF-1α (G and H). The images are representative from two patients out of total of 5 patients. The photomicrographs represent in situ immunohistochemistry performed on lung tissues. H and E staining of tissue obtained from transbronchial biopsy (I) 100X, HIF-1α immunostaining (J), negative staining using isotype control antibody (K). The brown color represents an area of precipitate formed by a chromogenic substrate that is transformed by an enzymatic label conjugated to the antibody that has bound to the HIF-1α antigen. Note that the intensity of the staining is most pronounced in the histiocytic cells (i.e., AMs and the multinucleated giant cells, thick arrow), and is not identified in the surrounding alveoli (thin arrow). The immunohistochemistry images are representative from one patient out of total of 5 patients.