Immunofluorescence and Confocal Microscopy of Human Tissues.

Tissue processing is detailed in SI Appendix. QRFP antibody (73) at 1:400 was applied overnight at room temperature, followed by a second incubation with QRFP antibody 1:400 and HCRT antibody 1:500 (goat HCRT anti HCRT, Santa Cruz Biotechnology, SC-8070) at 4 °C for 24 h. After rinsing with 1xPBS for 3 times (10 min each), secondary antibodies were applied (donkey IgGs coupled to Alexa-594, or -488 fluorophores) in 1:500 dilutions for 2 h at room temperature followed by rinsing with 1xPBS for 3 times (10 min each). To test the specificity of QRFP antibody, QRFP antibody was blocked with QRFP ({"type":"entrez-nucleotide","attrs":{"text":"NM_198180","term_id":"1653962193"}}NM_198180) Human Over-expression Lysate (2 μg/mL, 1%BSA) for 2 h at room temperature before use in the above-described immunofluorescence assay. Images were acquired on an inverted Zeiss LSM780 confocal laser-scanning microscope (405-, 488-, and 561-nm lasers) using a 40× oil objective (EC plan-Neofluar 40×/1.30 Oil DIC M 27). For each human section used for cell quantification, confocal images covering the HCRT positive cell field were acquired at 8-bit image depth and a frame of 1,024 × 1,024 pixels and tiled together at size of 2,125.48 × 2,125.48 μm using ZEN software. Immunoreactive cell counts were evaluated within that frame using ImageJ software.