To compare the size of hPSC-H15-derived NSPs treated with Dox versus no Dox treatment, for each independent experiment (n = 4), brightfield images were randomly taken for at least 3 NSPs for each condition using Zeiss Axio Observer z1 fluorescence microscope and ZEN imaging software. All images were taken at a consistent magnification for each experiment. ImageJ software was used to estimate the largest diameter of each NSP, briefly, by manually drawing a straight line across the centre of the NSP and the length of which was documented in μm. Statistical significance tests were performed using Mann–Whitney test.
Axio observer z1 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Axio Observer Z1 is a fluorescence microscope manufactured by Zeiss. It is designed for high-resolution imaging of fixed and live samples. The microscope features a motorized stage and automated functions to facilitate efficient data acquisition.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Apotome module, by Zeiss (3 mentions)
Calcein am dye, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Axiovision software, by Zeiss (3 mentions)
Hoechst, by Thermo Fisher Scientific (3 mentions)
Axiocam mrm camera, by Zeiss (2 mentions)
Apotome 2 extension, by Zeiss (2 mentions)
Tritc conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Axiovision zen 2012, by Zeiss (2 mentions)
Geltrextm ldev free reduced growth factor basement membrane matrix, by Thermo Fisher Scientific (2 mentions)
Zen software, by Zeiss (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
γh2ax antibody, by Merck Group (2 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
88 protocols using axio observer z1 fluorescence microscope
1
Measuring hPSC-H15-derived NSP Size
2
Immunofluorescence Labeling in Cultured Cochleae
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The cultured cochleae were processed for immunofluorescence with standard methods. Prior to the immunoreactions, all the cochleae were first treated for 30 min at room temperature with blocking solution (5% normal donkey and bovine serum in 0.05% Triton X-100 in PBS, pH 7.4). The cochleae were then incubated with the primary and secondary antibodies for each 2 h at room temperature. The used primary and secondary antibodies included: rabbit anti-Myosin7a (1:500, Proteus BioSciences, 25–6790), goat anti-Sox2 (1:300, Santa Cruz Biotechnology, sc-17320), rat anti-BrdU (1:2000, ABD Serotec, OBT0030CX), Alexa 647-conjugated donkey anti-rabbit IgG for Myosin7a labeling (cat. 711-606-152), DyLight 488-conjugated bovine anti-goat IgG for Sox2 labeling (cat. 805-545-180), and Alexa 594-conjugated donkey anti-rat IgG for BrdU labeling (cat. 712-585-150). The specificity of the antibodies has been declared in the protocols and proved in our preliminary experiments. All secondary antibodies were obtained from Jackson ImmunoResearch and diluted at 1:400. After the cochleae were sealed with Antifade Solution (Applygen), they were examined under an inverted Zeiss Axio Observer Z1 fluorescence microscope (20×) and an inverted Zeiss LSM700 laser-scanning confocal microscope (40×). Images were obtained using AxioVision 4.8 and Zen Light Edition programs.
3
Immunostaining of Cells and Tissue Sections
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For immunostaining of cells in culture, cells grown on glass coverslips (VWR) were washed in PBS, fixed in 4% paraformaldehyde in PBS, and permeabilized in 0.01% digitonin or 0.1% Triton X-100. For ear lesions from P80 Krt17+/+ or Krt17−/− Gli2tg/+ mice, tissues were fresh-frozen in Tissue-Tek O.C.T. (Sakura) and sections were cut at 5 µm. Samples were blocked in 5% normal goat serum (NGS) in PBS for at least 1 h before staining with primary antibodies diluted in blocking buffer for 16 h followed by Alexa Fluor 488– or Alexa Fluor 594–conjugated goat anti–mouse or goat anti–rabbit secondary antibodies (Invitrogen) for 1 h. 1 µg/ml of Hoechst 33342 was used to stain the nuclei, and coverslips were mounted on microscope slides with mounting media containing 1,4-diaza-bicyclo[2.2.2]octane (Electron Microscopy Sciences). Fluorescence images were obtained using the Zen Pro 2012 software (Carl Zeiss) with an Axio Observer.Z1 fluorescence microscope (Carl Zeiss) equipped with an AxioCam MRm camera (Carl Zeiss) at 37°C under a 63× Plan-Apochromat oil immersion lens (Carl Zeiss). The images were processed using Photoshop software (Adobe) to split and pseudo-color individual channels.
4
MRP2 Activity Quantification in HepaRG Cells
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The MRP2 activity was analyzed by incubation of the HepaRG cell layer in serum free Williams E medium without phenolred (GIBCO, Darmstadt, Germany) containing 5 μM 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDF-DA) (Sigma-Aldrich, Steinheim, Germany) or 10 μM 2-tert-Butyl-4-{(E)-3-[1-(5-carboxy-pentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-indol-(2Z)-ylidene]-propenyl}c-7-diethylamino-1-benzopyranylium (DY-635) (Dyomics, Jena, Germany) at 37°C. After 15 min the cell layer was incubated with William’s medium E without phenolred for 30 min. Subsequently, cells were analyzed using an AXIO Observer Z1 fluorescence microscope with Apotome 2 extension (Carl Zeiss AG; Jena, Germany). The software FiJi (GNU General Public License) was used for image analysis.
5
Quantitative Analysis of Tau-Tubulin Interactions
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Most imaging was done using an Axio Observer Z1 fluorescence microscope (Zeiss) at 40X magnification, digitally zoomed 4 times. All images were collected using the AlexaFluor488 (EGFP), AlexaFluor594 (secondary antibodies), and UV (DAPI) settings. For each condition in every experiment, 10 images were taken randomly from 2 independently transfected and stained sets of cells. For analyzing tau-tubulin interactions, the images were screened for rings and the number of cells with them was counted. For analysis of the nuclear membrane, images were processed in ImageJ to select for the nuclei of tau-positive cells. The mean fluorescence intensity, roundness, and area of the nucleus were recorded. The nuclear area factor was calculated by multiplying the area and roundness of each nucleus. The high magnification (63 × 3) images in Figure 1B were obtained on a Leica SP2 AOBS Confocal Microscope. Z-stacks of these images were imported into IMARIS software for 3D reconstruction.
6
Comet Assay for DNA Damage Analysis
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Neutral and alkaline comet assays were performed according to the manufacturer’s protocol (Trevigen) by performing electrophoresis at 4 °C. Slides were visualized with a AxioObserver Z1 fluorescence microscope (ZEISS) with the objective EC Plan-Neofluar 10X/0.3 Ph1. Comet tail moments were analyzed with ImageJ software (version 1.52p) automatically with the plugin OpenComet (http://www.cometbio.org ) or manually using a macro provided by Robert Bagnell (https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay ) as previously described70 (link). Data were represented by using box-and-whisker plot with GraphPad Prism 8.3.1 or 9.1.0 software with the following settings: boxes: 25–75 percentile range; whiskers: 10–90 percentile range; horizontal bars: median. In Supplementary Fig. 8c , data were represented by using scattered dot blot with the horizontal bar indicating the mean.
7
Immunostaining Analysis of Frozen Tissue Samples
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Histological analysis was performed on snap frozen tissue. Tissue sections were fixed in acetone or 10% neutral buffered formalin, blocked with 5% FCS/0.3% Triton-X in PBS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8, cleaved Caspase-9, p-CREB (Ser 133), p-AMPK (Thr172) (all from Cell Signaling), CD8 and MHC-I (both from BD Biosciences) followed by incubation with the appropriate secondary antibodies. Cy3-conjugated anti-rabbit secondary antibodies were used for immunofluorescence. HRP-linked anti-rabbit secondary antibodies were used for conventional staining, which were visualised with the Peroxidase Substrate (ImmPACT NovaRED). Images were taken with an Axio Observer Z1 fluorescence microscope or Axiocam 503 color microscope (ZEISS) and quantified using Image J using the fluorescence intensity (MFI) per area as previously described [72 (link)].
8
Immunofluorescence Staining of HFF Cells
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HFF cells grown on coverslips in six-well dishes (3x105 cells/well) were washed twice with PBS at indicated times. Cells were fixed with a 4% paraformaldehyde solution for 10 min at room temperature (RT) and then washed for two times. Permeabilization of cells was achieved by incubation with 0.2% Triton X-100 in PBS on ice for 20 min. Cells were washed again with PBS over a time period of 5 min and incubated with the appropriate primary antibody diluted in PBS-1% FCS for 30 min at 37°C. Excessive antibodies were removed by washing four times with PBS, followed by incubation with the corresponding fluorescence-coupled secondary antibody diluted in PBS-1% FCS for 30 min at 37°C. The cells were mounted using the DAPI-containing Vectashield mounting medium (VECTOR LABORATORIES, Burlingame, CA, USA) and analyzed using either a Leica TCS SP5 confocal microscope with the 543-nm laser line or an Axio-Observer.Z1 fluorescence microscope (Carl Zeiss Microscopy GmbH) with 469/38 nm, 555/30 nm, 631/33 nm LED sources.
9
miRNA-224 FISH Localization in Mouse Testis
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Mmu‐miR‐224 detection probe, scrambled probe and detection kits were purchased from Focobio Corporation (Guangzhou, China). The fluorescence in situ hybridization (FISH) assay was performed as previously described 16 . Briefly, mouse testicular tissue and adult testis were fixed and embedded in paraffin. The slides (5 μm) were dewaxed, incubated in solution A and solution B for 20 min. and 15 min. respectively. After that, the slides were washed and fixed in 4% formaldehyde for 15 min. Before pre‐hybridization with solution C, the slides were washed in PBS for 10 min. The slides were hybridized by adding 10 μl hybridization solution containing 1.5–2.0 μM miR‐224 probe overnight in the 40–42°C incubator. In the second day, the slides were washed in washing buffer I for 15 min. then rinsed in washing buffer II twice for 15 min. each. The slides were washed in 75%, 100% ethanol for 2 min., respectively, and air‐dried for 10 min., which was followed by adding 10 μl DAPI for 10 min. The slides could be analysed by Axio Observer Z1 fluorescence microscope (Zeiss, Germany).
10
Immunofluorescence Analysis of cGAS and γH2A.X
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