Fig 1: IHC of salivary gland for the detection of ACE2 and TMPRSS2 complex. (a) IHC of parotid glands for the detection of ACE2. The brownish-yellow ACE2 immune complex was observed in the cytoplasm and cytomembrane of epithelial cells from serous acinar, sacral, secretory, excretory ducts, and vascular endothelial. (b) IHC of parotid glands for the detection of TMPRSS2 in parotid glands. The brownish-yellow TMPRSS2 immune complex was observed in the cytoplasm and cytomembrane of epithelial cells from serous acinar, sacral, secretory, excretory ducts, and vascular endothelial. (c) IHC of submandibular glands for the detection of ACE2. The brownish-yellow ACE2 immune-complex was mainly observed in the cytoplasm and cytomembrane of duct epithelial cells and serous acinus cells; the mucous acinar cells were negatively immunostained for the ACE2 immune complex. (d) IHC of submandibular glands for the detection of TMPRSS2. The brownish-yellow TMPRSS2 immune-complex was mainly observed in the cytoplasm and cytomembrane of duct epithelial cells and serous acinus cells; the mucous acinar cells were negatively immunostained for the TMPRSS2 immune complex. (e) IHC of sublingual glands for the ACE2 detection. The mucous acinar cells were found to be negatively immunostained for the brownish-yellow ACE2 immune complex; the ACE2 immune complex was observed in the cytoplasm and cytomembrane of serous acinar cells in the mixed acinus. (f) IHC of sublingual glands for the detection of TMPRSS2. The mucous acinar cells were found to be negatively immunostained for the brownish-yellow TMPRSS2 immune complex, which was observed in the cytoplasm and cytomembrane of serous acinar cells in the mixed acinus (black scale bar = 100 µm; black arrow: secretory duct; blue arrow: sacral ducts; yellow arrow: excretory ducts; red arrow: serous acinar cells) [Colour figure can be viewed at wileyonlinelibrary.com]
Fig 2: The exogenous ACE2 and TMPRSS2 are absorbed to oral mucosa epithelial cells. (a) The results of the western blot confirmed that exogenous ACE2 was detected in HOEC and HOK cells. (b) The results of western blot confirmed that exogenous TMPRSS2 was detected in HOEC and HOK cells. (c) His-tag was positive in HEK-293T transfected with a His-tagged Staphylococcus aureus cas9 and negative in HEK-293T; HOEC and HOK, only stained by secondary antibodies, were negative. (d) The results of ICC confirmed that exogenous ACE2 was observed in the cytomembranes of HOEC and HOK cells. (e) The results of ICC confirmed that exogenous TMPRSS2 was observed in the cytomembranes of HOEC and HOK cells. (black scale bar = 100 µm) [Colour figure can be viewed at wileyonlinelibrary.com]
Fig 3: The ACE2 and TMPRSS2 expression in salivary glands. (a) The ACE2 expression levels in parotid glands, submandibular glands, and sublingual glands, in descending order. (b) The TMPRSS2 expression levels in parotid glands, submandibular glands, and sublingual glands, in descending order. (c) The blots of ACE2 and TMPRSS2. (d)The controls in western blot. ACE2 was positive in small intestine and negative in breast tissue; TMPRSS2 was positive in prostate adenocarcinoma and negative in adipose tissue; and salivary glands only stained by the secondary antibody were negative
Fig 4: The binding of SARS-CoV-2 spike protein with ACE2 in human salivary glands demonstrated by the ELISA. (a) The binding of SARS-CoV-2 spike protein with ACE2 in parotid glands. (b) The binding of SARS-CoV-2 spike protein with ACE2 in submandibular glands. (c) The binding of SARS-CoV-2 spike protein with ACE2 in sublingual glands. (d) The spike protein of SARS-CoV-2 was positively absorbed to the homogenate of small intestine. (e) The spike protein of SARS-CoV-2 was negatively absorbed to the homogenate of breast tissue
Fig 5: The controls for ACE2 and TMPRSS2 in IHC. (a) H and E staining of the small intestine. (b) The positive controls for ACE2: The brownish-yellow ACE2 immune complex was observed in the small intestinal epithelium. (c) H and E staining of the breast tissue. (d) The negative controls for ACE2: The brownish-yellow immune complex was not observed in the breast tissue. (e) H and E staining of prostate adenocarcinoma. (f) The positive controls for TMPRSS2: The brownish-yellow TMPRSS2 immune complex was observed in the prostate adenocarcinoma. (g) H and E staining of adipose tissue. (h) The negative controls for TMPRSS2: The brownish-yellow immune complex was not observed in the adipose tissue. (i) The controls without primary antibodies for ACE2 and TMPRSS2: The salivary glands were found to be negatively immunostaining. (j) The isotype controls for ACE2 and TMPRSS2 (normal rabbit IgG): The salivary glands were found to be negatively immunostaining. (black scale bar = 100 µm) [Colour figure can be viewed at wileyonlinelibrary.com]
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