Zen 2011 lsm 700 software

With the help of a Zeiss LSM DUO confocal lasers scanning microscope with a META module, sections were analyzed, and pictures were taken (Carl Zeiss MicroImaging GmbH, Jena, Germany). Two helium–neon lasers (543 and 633 l) and two argon lasers (458 and 488 l) are included in this microscope [25 (link)]. A 2048 by 2048 pixel array with an 8-bit resolution was created by digitizing each image. Using helium–neon (543 nm) and argon (458 nm) lasers with a scanning speed of 1 min and 2 s and up to eight averages, optical slices of fluorescence samples were obtained. The images were refined using Zen 2011 (LSM 700 Zeiss software, Oberkochen, Germany). Each picture was quickly taken to prevent photo degradation. Digital photo cropping was conducted in Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA) to build the figure montage [21 (link)]. The intensity profile of an image was displayed on a freely selected line using the “display profile” feature of the laser scanning microscope. The intensity curves are presented alongside the scanned images in graphs.

Sections were analyzed and images acquired using a Zeiss LSM DUO confocal laser scanning microscope with META module (Carl Zeiss MicroImaging GmbH, Jena, Germany) equipped with an argon laser (458, 488 l) and 2 helium-neon lasers (543 and 633 l). All pictures were digitalized at an 8-bit resolution into a 2048 × 2048-pixel array. Optical slices of fluorescence samples were acquired using a 1-min, 2-s scanning speed and up to 8 averages using a helium-neon laser (543 nm) and an argon laser (458 nm). We obtained 1.50-µm-thick sections using a pinhole of 250; the images captured were processed using Zen 2011 (LSM 700 Zeiss software). To reduce photodegradation, each image was captured quickly. Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA) was used to crop digital pictures and create the figure montage. The “display profile” function of the laser scanning microscope was used to show the intensity profile on an image along a freely selectable line. The intensity curves are shown in the graphs next to the scanned images.

Slices received treatment in a 2.5% solution of bovine serum albumin (BSA) after deparaffinization and rehydration. After that, primary anti-TLR2 and anti-iNOS antibodies were incubated in the sections. The incubation of secondary antibodies was carried out after one night of exposition to the primary antibodies. To prevent photobleaching, the slices were mounted using Fluoromount [40 (link),41 (link)]. As a negative control, experiments were carried out without the primary antibodies. Rat gut tissues were used as a positive control to ensure the immunopositivity of the primary antibodies (data not shown). The slices were examined using a confocal laser scanning microscope with a META module (Zeiss LSM DUO, Carl Zeiss MicroImaging GmbH, Jena, Germany). The fluorescence was detected using argon (458 nm) and helium–neon (543 nm) lasers. The images were captured and enhanced using Zen 2011 (LSM 700 Zeiss software, Oberkochen, Germany). To prevent photodegradation, each picture was snapped as rapidly as possible. The digital pictures were added to a figure composite using Adobe Photoshop CC version 2019 (Adobe Systems, San Jose, CA, USA). The fluorescence intensity curves were then evaluated using Zen 2011 “Display profile” feature. Information about the antibodies is provided in Table 1.