Postnatal and adult ovaries were collected, fixed in 4% paraformaldehyde, and paraffin-embedded. Then, 4-µm sections were cut and processed for histology and immunofluorescence. Sections were stained with periodic acid-Schiff (PAS) stain using standard protocols. Immunofluorescence was performed as previously described103 (link). Overnight incubation with primary antibodies at 4 °C was followed by incubation with the appropriate secondary antibodies (1/800) (Alexa-Ig, Molecular Probe). Nuclei were stained with DAPI (Sigma). Images were captured with a Zeiss Axioimager Apotome microscope. For quantification of SUMO1 and SUMO2 immunofluorescence signals, images were captured with a Zeiss LSM780 confocal microscope and analysed with the Imaris V9.5. Statistical analyses were performed using the GraphPad Prism v9 software using two-way ANOVA with Dunnett’s multiple comparisons test.
Axioimager apotome microscope
Manufactured by Zeiss
Sourced in Germany
The Axioimager Apotome microscope is a high-performance imaging system designed for advanced microscopy applications. It combines a structured illumination technique, known as Apotome, with the precision and reliability of the Axioimager platform. The core function of this microscope is to enable optical sectioning and improve image contrast, allowing for enhanced visualization of fine structural details in samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (3 mentions)
Apotome microscope, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Carl Roth (2 mentions)
Zen 2.0 lite software, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bx52 microscope, by Olympus (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
B0586, by Agilent Technologies (1 mentions)
Mab1561, by R&D Systems (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cd45 2d1, by BD (1 mentions)
Axiovision imaging software, by Zeiss (1 mentions)
γh2ax, by Merck Group (1 mentions)
Alexafluor 647 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexafluor 555 conjugated anti rabbit, by Abcam (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
13 protocols using axioimager apotome microscope
1
Histological and Immunofluorescence Analysis of Postnatal and Adult Ovaries
2
Multistep Immunofluorescence Staining Protocol
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A multistep staining protocol was designed based on the characteristics of the primary antibodies used. Briefly, the slides were hydrated, permeabilized with 0.5% TritonX-100 in PBS for 10 min, followed by blocking with either 10% goat or donkey antiserum in PBS (depending on the secondary antibody used) for 1 h. Thereafter, the slides were washed several times and incubated with the primary antibody in blocking solution for 1 h at room temperature. Tissue sections were then rinsed several times with PBS and incubated for 1 h at room temperature with a secondary antibody against the respective primary antibody. A similar procedure was followed for all antibodies in a sequence procedure. Then the slides were rinsed, and the cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, 2 drops/ml according to the manufacturer’s protocol), mounted with mounting medium (ProLongTM Gold Antifade mountant, Invitrogen, Grand Island, NY), and images were captured digitally using the Axio Imager Apotome microscope (Zeiss, Göttingen, Germany). A minimum of 20 fields at 63X magnification per case was used to evaluate for the presence of neutrophils (CD11b). Similarly, the presence of NE/MPO/H3Cit triple staining in which nuclear morphology and NETs were determined.
3
Immunofluorescence Staining of Ovarian Somatic Cells
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OSCs were plated on concanavalin A-coated coverslips, fixed with 4% paraformaldehyde in PBS (RT, 10 min), washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS (15 min), and blocked with 2% bovine serum albumin, 0.02% Tween 20 in PBS. Primary antibodies were against Piwi G-1 (1:500, mouse, Santa Cruz sc-390946) and MEP-1 (1:500, Rabbit, A. Brehm). Nuclei were stained with DAPI (Sigma). Images were captured with a Zeiss Axioimager Apotome microscope.
4
Quantifying Hippocampal Cell Populations
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Briefly, images were collected with a Zeiss AxioImager Apotome microscope using a 20× objective. The parameters were kept constant across sections. Regions of interest were selected using Zeiss imaging software (Carl Zeiss, Hertfordshire, UK) and the numbers of positive cells were counted from both the upper and lower blades of the dentate gyrus of both hippocampi. Manual counts of were performed by an experimenter blind to the experimental conditions. At least four sections per animal were analyzed, from the medial portion of the dorsal hippocampus (from 3.2 to 4.00 mm posterior to bregma). For each section cells were counted (without knowledge of treatments) under high power (×40) using an Apotome Zeiss microscope (Carl Zeiss, Hertfordshire, UK).
5
Microscopic Image Analysis Protocol
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Slides stained for histology or immunohistochemistry were either imaged using the Olympus BX52 microscope (Olympus America Inc., Allentown, PA, USA) or using the AxioImager Apotome microscope (Zeiss, Jena, Germany). Image analysis was performed using ImageJ software. Analyses parameters in ImageJ were maintained constant for every staining in all the slides, in order to avoid bias and exclude background interference.
6
Quantifying Synaptic Vesicle Proteins in C. elegans
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Young adult animals carrying the juIs1 [unc-25p::snb-1::GFP + lin-15 (+)] IV transgene in a wild type or lin-12 (n137 ) background were paralyzed in 30mg.mL-1 2,3-Butanedione monoxime (BDM, Sigma B0753) in M9 and mounted on 2% agar pads. Z-stack (distance between images: 0.2μm) images of SNB-1::GFP puncta were taken from the ventral nerve cord between motor neurons VD10 and VD11, using the Zeiss AxioImager ApoTome microscope at 100X magnification. Images formed by merging 3 layers were analyzed using the ‘punctaanalyser” MATLAB program from (Kim et al. 2008 (link)). Results from control and experimental animals were tested for significance using Student’s t-test.
7
Nuclei Quality Assessment via DAPI Staining
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For picture acquisition and to check the nuclei quality, nuclei were DAPI-stained (24 (link)). All pictures were taken in the DAPI and differential interference contrast (DIC) channels using the Zeiss Axioimager Apotome microscope equipped with Zeiss AxioVision software.
8
Visualizing Sperm Nucleus Transformation
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Sperm nuclei incubated in X. laevis egg extracts were supplemented with Cy3-dCTP and 2 μl of this solution was mounted between slide and coverslip in fixing solution (45 mM PIPES pH 7.2, 45 mM NaCl, 240 mM KCl, 10% formalin, 50% glycerol, 2 μg ml−1 Hoechst and 3,3′-dihexyloxacarbocyanine (DiOC6)) to visualise DNA and vesicle membrane fusion. Slides were analysed by fluorescence microscopy. Immunofluorescence staining was performed as follows. The reaction mixture was diluted 10 times in XB buffer, and nuclei were spun on coverslips through a 0.5 M sucrose cushion at 100×g for 10 min. Nuclei on coverslips were fixed in 4% paraformaldehyde for 10 min and then washed with PBS/0.1% Tween 20 before blocking with 3% BSA in PBS/0.1% Tween 20. Incubation with primary antibodies was performed at 4 °C and overnight, while incubation with secondary antibodies was performed at room temperature (RT) for 1 h. ProLong Gold (Thermo Fisher Scientific) was used for slide mounting. A Zeiss Axioimager ApoTome microscope was used for image acquisition and ImageJ for image analysis.
9
Immunohistochemical Analysis of Liver Cryosections
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Liver cryostat sections were prepared as previously described22 (link) and immunostained with antibodies against HBc (B0586 [Dako, Glostrup, Denmark] or C1-5 [Santa Cruz, Dallas, TX]), and human antigens: albumin (A0001 [Dako] or A80-129A [Bethyl]), CD3 (A0452 [Dako] or UCHT1 [eBiosciences]), CD45 (2D1 [BD Biosciences]), CD68 (KP1 [Dako]), CK7 (M7018 [Dako]) EpCAM (OP187 [Calbiochem, San Diego, CA]), PD-L1 (MAB1561 [R&D Systems]). Secondary antibodies were coupled to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (Molecular Probes, Eugene, OR). Photomicrographs were taken with an Axioimager Apotome microscope (Zeiss, Oberkochen, Germany).
10
Quantifying Hippocampal Newborn Cells
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