Lsm duo

Deparaffinized and rehydrated slices were treated with bovine serum albumin (BSA) (2.5%). Then, the sections were exposed to primary antibodies against MAPK p38, TLR2, Piscidin1, 5-HT, and iNOS [28 (link)]. Subsequently, each section was assessed separately and in double-label tests. Then secondary antibodies were incubated. To prevent photobleaching, the sections were mounted with Vectashield (Vector Labs, Burlingame, CA, USA). As a negative control, experiments were run without the primary antibodies. Rat skin tissues were used as a positive control to ensure the primary antibodies’ immunopositivity [29 (link),30 (link)].
Slices were evaluated by a confocal laser scanning microscope (Zeiss LSM DUO, Carl Zeiss MicroImaging GmbH, Jena, Germany, Europe) with a META module. Optical slices of fluorescence samples were generated by two types of lasers: helium-neon (543 nm) and argon (458 nm). The scanning rate was 62 s. The images were improved with Zen 2011 (LSM 700 Zeiss software Oberkochen, Germany, Europe). To avoid photo deterioration, each picture was snapped as rapidly as possible. To create the figure composite, a digital photo was edited using Adobe Photoshop CC ver. 2019 (Adobe Systems, San Jose, CA, USA). The “display profile” function of Zen 2011 was then used to evaluate the intensity curves of fluorescence. The information about antibodies is enclosed in Table 1.

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.

Cells grown on poly-l-lysine-coated coverslips were fixed in 4% paraformaldehyde in PBS for 15 min, treated with 50 mm NH₄Cl in PBS for 10 min, permeabilized in 0.5% Triton X-100 in PBS for 15 min, and blocked with 2% bovine serum albumin in PBS. Incubations with rabbit polyclonal anti-FOXO3a 1:200 (1112; Cemines, epitope SADDSPSQLSKWPGS) and Alexa 488- or Alexa 546-conjugated goat anti-rabbit IgG 1:2000 (Molecular Probes) antibodies were carried out in 0.2% BSA and 0.1% Tween 20 in PBS at room temperature for 1.5 h each. The samples were mounted in ProLong Gold antifade reagent with 4′-6-diamidino-2-phenylindole (Molecular Probes) and analyzed by confocal microscopy (LSM Duo; Zeiss).