Tsc sp5

For localization analysis of receptors and neuropeptides by confocal laser scanning microscopy (CLSM), yeast cells were transfected with GFP fusion constructs and stained with the membrane specific dye Dil (Merck KGaA, Germany). In brief, cells were fixed for 10 min on ice with 4% paraformaldehyde dissolved in PBS. After two washing steps, cells were incubated with 10 μm Dil and washed four times with PBS. Finally, yeasts were dried and mounted with Roti^®-Mount FluorCare (Carl Roth, Germany). Stained yeast cells were captured on an inverse CLSM (Leica TSC SP5; Leica, Germany). GFP was excited with an argon-ion laser at 488 nm and Dil at 561 nm.

Human kidney samples were embedded in optimal cutting temperature (OCT) medium and ultrathin cryosections (5 μm) placed on Superfrost/Plus Microscope Slides (www.fishersci.com, accessed on 22 November 2022). Human kidney cryosections were fixed with methanol at −20 °C for 10 min, treated with SDS 1% for 10 min and subjected to immunofluorescence analysis. Nonspecific binding sites were blocked with 1% bovine serum albumin in PBS for 30 min. Sections were then incubated with the primary antibodies rabbit anti-BAR3 (SC-50436, Santa Cruz Biotechnology, Inc., www.scbt.com, accessed on 22 November 2022) with mouse anti-AQP2 antibody [38 (link)] (SC-515770, Santa Cruz Biotechnology, Inc., www.scbt.com, accessed on 22 November 2022) or mouse anti-NKCC2 [20 (link)] or mouse anti-AQP1 [39 (link),40 (link)] (SC-25287, Santa Cruz Biotechnology, Inc., www.scbt.com, accessed on 22 November 2022). Sections were incubated with Alexa Fluor-conjugated secondary antibody. Confocal images were obtained with a confocal laser-scanning microscope (Leica TSC-SP5, Leica, www.leica-microsystems.com, accessed on 22 November 2022).

For morphological analyses by CLSM, worms were fixed in AFA (95% ethanol, 3% formaldehyde, and 2% glacial acetic acid) for at least 24 h at 4°C. After staining with Certistain carmine red (Merck, 1390-65-4), as previously described (92 (link), 93 (link)), worms were destained in acidic 70% ethanol and dehydrated progressively in 90% and 100% ethanol. Worms were mounted on glass slides with Canada balsam (Sigma-Aldrich, 03984). A TSC SP5 inverse confocal laser scanning microscope (Leica, Germany) was used for imaging. Carmine red was excited using an argon-ion laser at 488 nm. For fluorescence in situ hybridization, samples were imaged on an inverse CLSM. Cy3 and Cy5 were excited with 561 and 633 nm, respectively.
For EdU labeling and detection of proliferating cells, the Click-iT Plus EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) was used. After 24 h of incubation with EdU, couples were separated, fixed, and stained as described (94 (link)). Worms were counterstained with 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst 33342) in a final concentration of 8 µM. Stained worms were examined on an inverse CLSM (Leica TSC SP5; Leica, Germany). Hoechst was excited with a 405-nm laser and Alexafluor488 with an argon-ion laser at 488 nm.

Caco-2 cell monolayers grown on 0.33 cm² Transwell supports were fixed with 4% paraformaldehyde in PBS for 30 min, washed, and permeabilized in 0.3% Triton X-100 in PBS. Then, they were incubated with a monoclonal antibody against Zonula occludens-1 (ZO-1; Invitrogen, Carlsbad, CA, USA), washed, and incubated with FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) and DAPI. Stained monolayers were examined using a Leica TSC SP5 (Leica Microsystems, Wetzlar, Germany) confocal microscope equipped with × 60 objectives. Stacks were imported into AutoVisualize 9 (AutoQuant Imaging) for presentation of 3D projections and maximum volume projections were created with the 5D viewer deconvolution software.

Coverslip cultures were fixed in 4% paraformaldehyde for 10 min at room temperature. After adequate washing with PBS, cells were incubated in a blocking buffer (10% donkey serum plus 0.2% Triton X-100 in PBS) for 60 min at room temperature followed by primary antibody incubation at 4 ^oC overnight. On the next day, coverslips were washed with PBS and stained with the fluorescently conjugated secondary antibodies (1:1000, Jackson, West Grove, PA). Nuclei were counterstained with Hoechst 33258. Coverslips were visualized with Leica TSC SP5 (Leica Microsystems, Bensheim, Germany) confocal laser-scanning microscope. Antibodies used in this study included Pax6 (1:1000, rabbit IgG, Covance), Nkx2.1 (1:400, mouse IgG, Chemicon), FoxG1 (1:1000, rabbit IgG, Abcam), Sox1 (1:500, goat IgG, R&D), Sox2 (1:1000, goat IgG, R&D), Oct4 (1:1000, mouse lgG, Santa Cruz), HoxB4 (1:50, mouse IgG, DSHB), Nestin (1:500, mouse IgG, millipore), Map2 (1:10,000, chicken IgY, abcam), Tuj1 (1:5000, mouse IgG, sigma), cleaved caspase 3 (1:500, rabbit IgG, CST), BrdU (1:200, rat IgG, Abcam), Ki67 (1:500, rabbit IgG, Abcam), pH3 (1:500, rabbit IgG, CST) and ZIKV (1:500, mouse anti-flavivirus group antigen antibody, millipore). Light images were visualized with Leica DMI3000 (Leica Microsystems, Bensheim, Germany) microscope and organoid size was measured with Leica application Suite softwares.

Spinal cord slices were fixed with 4% paraformaldehyde at room temperature for 1 h. Slices were then washed twice with phosphate-buffered saline (PBS) for 15 min, blocked with 5% normal horse serum and 0.2% Triton-X100 in PBS, and incubated overnight at 4 °C with antibody against mouse anti-neurofilament heavy-chain (NF-H) (SMI-32, 1:1000) (Sternberger Monoclonals Inc.; Baltimore, MD, USA). Cultures were then thoroughly washed in PBS with 0.2% Tween-20 (PBS-T) and incubated with appropriate secondary antibody Alexa Fluor^®488 goat anti-mouse IgG (1:1000) diluted in blocking buffer for 1 h at room temperature. Then, slices were washed two times with PBS-T, incubated for 20 min with DAPI (4′,6-Diamidino-2-phenylindole), diluted in PBS, and washed several times. Finally, slices were mounted in Superfrost^®Plus slides (Thermo Fisher Scientific; Waltham, MA, USA) with Fluoromount-G mounting medium (SouthernBiotech; Birmingham, AL, USA) and fluorescence was visualized under an epifluorescence microscope (Nikon Eclipse 90i, Nikon Instruments Inc.; Melville, NY, USA) or a confocal microscope (Leica TSC SP5, Leica Microsystems; Deerfield, IL, USA). A minimum of 15 sections were used for MN counting for each experimental condition.