Journal: STAR Protocols

Article Title: Imaging of Telomerase RNA by Single-Molecule Inexpensive FISH Combined with Immunofluorescence

doi: 10.1016/j.xpro.2020.100104

Figure Lengend Snippet: Super-Resolution Imaging of hTR smiFISH Combined with IF against Coilin and TRF2 (A) HeLa 1.3 cells hybridized with the 15 probes set for hTR smiFISH labeled with FLAP-Y-Cy5 shown in magenta. (B) Coilin (in blue) and TRF2 (in green) were detected by immunofluorescence. (C) Overlay of coilin, TRF2, and hTR smiFISH. (D) Overlay of hTR with DNA stain in gray, scale bar, 10 μm. (E–L) Zoom on regions of image C showing Cajal bodies (E–H) or telomeres (I–L) with partially overlapped hTR signal. Panels (E–H) scale bars, 0.5 μm. Panels (I–L) scale bars, 0.2 μm. Maximum intensity projection of Z stacks acquired on a Zeiss Elyra PS.1 super-resolution microscope by structured illumination using an Andor iXon3 EMCCD camera (resolution after SR-SIM processing x, y = 0.040 μm).

Article Snippet: Maximum intensity projection of Z stacks acquired on a Zeiss Elyra PS.1 super-resolution microscope by structured illumination using an Andor iXon3 EMCCD camera (resolution after SR-SIM processing x, y = 0.040 μm).

Techniques: Imaging, Labeling, Immunofluorescence, Staining, Super-Resolution Microscopy

Journal: The Journal of Infectious Diseases

Article Title: Characterization of p57, a Stage-Specific Antigen of Pneumocystis murina

doi: 10.1093/infdis/jiy099

Figure Lengend Snippet: Anti-rp57 serum reacts with a subset of Pneumocystis murina organisms, as demonstrated by immunofluorescence microscopy. A, P. murina–infected CD40L knockout (KO) mouse lung tissue sections were dual labeled using anti-p57 (green) and anti–β-1,3 glucan (magenta) antibodies. p57 is not expressed on the surface of cysts, which are identified by the anti–β-1,3 glucan antibody, but is expressed on intracystic bodies of some cysts (inset). p57 is also expressed on multiple small organisms that are presumably trophic forms. The broken line box outlines the cyst magnified in the inset, where p57 antibody labeling of the intracystic bodies is apparent. The main and inset images are both maximum projections of a 5-µm-thick z-stack imaged on a Nikon 90i epifluorescence microscope and deconvolved. B, Labeling multiple life stages of Pneumocystis, using anti–major surface glycoprotein (Msg) antibody (red) and anti-p57 (green). Although Msg is present on both cysts and trophic forms, p57 and Msg expression appear to be largely mutually exclusive. p57 is expressed by smaller organisms, while Msg is expressed by larger trophic forms, as well as cysts. The broken line box outlines a cyst (based on morphology) that is magnified in the inset as a single slice from the same z-stack, again demonstrating labeling of intracystic bodies but not the cyst surface. The main image is a maximum projection of an 8-µm-thick z-stack imaged on a Leica SP8 confocal microscope. All images have DAPI (blue). The bars denote 10 µm in the main images and 2 µm in the insets. A more detailed figure showing individual labeling for all markers, as well as a merge, is provided in the Supplementary Data.

Article Snippet: All images are maximum projections of approximately 6-µm-thick z-stacks imaged on the Nikon 90i microscope and deconvolved.

Techniques: Immunofluorescence, Microscopy, Infection, Knock-Out, Labeling, Antibody Labeling, Expressing

Journal: The Journal of Infectious Diseases

Article Title: Characterization of p57, a Stage-Specific Antigen of Pneumocystis murina

doi: 10.1093/infdis/jiy099

Figure Lengend Snippet: Immunofluorescence microscopy of Pneumocystis murina–infected CD40L knockout (KO) and C57BL/6 mouse lung tissue. All sections were labeled with anti-p57 antibody (green), anti–major surface glycoprotein antibody (red), and anti–β-1,3 glucan (magenta). A, Lung section from a CD40L KO mouse treated with caspofungin for 21 days. Cysts are largely eliminated, and, in conjunction with that, there is very limited expression of p57. B, Lung section from a P. murina–infected C57BL/6 mouse 28 days after the start of cohousing. Although the peak level of infection is typically 2–3 log10 less than in CD40L KO mice, p57 expression is easily identified. C, Lung section from a C57BL/6 mouse immunized with p57-pMALHis and subsequently infected with P. murina. p57 expression is again easily identified, and the level of infection in immunized animals is similar that in unimmunized animals (see also Figure 7). All images are maximum projections of approximately 6-µm-thick z-stacks imaged on the Nikon 90i microscope and deconvolved. The bar is 10 µm. A more detailed figure showing individual labeling for all markers and DAPI, as well as a merge, is provided in the Supplementary Data.

Article Snippet: All images are maximum projections of approximately 6-µm-thick z-stacks imaged on the Nikon 90i microscope and deconvolved.

Techniques: Immunofluorescence, Microscopy, Infection, Knock-Out, Labeling, Expressing