In brief, we used the One Step TUNEL Apoptosis Assay Kit (Beyotime, C1088) according to the instruction provided by the manufacturer. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in gradient ethanol (100%, 100%, 95%, 80%, 75%). Proteinase K was diluted with 10 mmol/L Tris-HCl (1:1000), then sections were incubated at RT for 30 min and rinsed with PBS. Next, the sections were stained with TUNEL working solution at 37°C for 1 h, followed by incubation with Hoechst 33342 (Thermo Fisher, H3570, 1:1000) to visualize the nucleus. Finally, the sections were mounted in VECTERSHIELD® anti-fading mounting medium (Vector Laboratories, h-1000), and images were obtained using Zeiss LSM900 confocal system. Image J was used to quantify the percentage of TUNEL-positive cells.
Lsm900 confocal system
Manufactured by Zeiss
Sourced in Germany
The ZEISS LSM 900 is a confocal laser scanning microscope system that enables high-resolution imaging of samples. It provides optical sectioning capabilities, allowing for the acquisition of 3D image data. The LSM 900 utilizes a laser light source and specialized optics to achieve detailed visualization of biological and materials science specimens.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
One step tunel apoptosis assay kit, by Beyotime (2 mentions)
Vectershield anti fading mounting medium, by Vector Laboratories (1 mentions)
Mitotracker red probe, by Thermo Fisher Scientific (1 mentions)
Triolein, by Merck Group (1 mentions)
Antifade mounting medium, by Vector Laboratories (1 mentions)
Anti sqstm1, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Cell imaging dish, by NEST Biotechnology (1 mentions)
Goat serum, by Boster Bio (1 mentions)
Alexa fluor 555 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti p chek1, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Sa β gal, by Dojindo Laboratories (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
10 protocols using lsm900 confocal system
1
Quantifying Apoptosis via TUNEL Assay
2
Mitochondrial Imaging in Fibroblasts
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Fibroblast cells were cultured in glass‐bottomed dishes. The mitochondria was visualized using MitoTracker red probe (Invitrogen). Then cells were washed with ice‐cold phosphate‐buffered saline (PBS) three times and were fixed with 4% paraformaldehyde for 15 min at room temperature. Fluorescence images were captured by ZEISS LSM900 confocal system.
3
PLIN1 AH Peptide Binding Dynamics
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PLIN1 AH peptides (human PLIN1 108–143: PPEKIASELKDTISTRLRSARNSISVPIASTSDKVL, FITC-modified or unmodified) were synthesized by ThermoFisher and dissolved in the emulsion buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) at a concentration of 1 mg/mL. Phospholipid (L-a-Phosphatidylcholine soybean, P7443, Sigma) was dissolved in chloroform at a concentration of 32 mM as a stock solution. A 5 μL aliquot of the phospholipid stock solution was dried under nitrogen stream in a 1.5 mL microcentrifuge tube, followed by the addition of 90 μL emulsion buffer, 5 μL Triolein (>99% purify, T7140 Sigma), and 5 μL PLIN1-AH-FITC. For neat oil droplets, 90 μL emulsion buffer was mixed with 5 μL Triolein and 5 μL PLIN1-AH-FITC in a clean 1.5 ml microcentrifuge tube. The tubes were then vortexed manually at a fixed angle of ~30° for 10 cycles of 30 s on/30 s off at 25°C and sonicated in a Bioruptor® Pico sonication device (B01080010) for 30s at a medium frequency level to allow emulsion to happen. To start the competition assay, the emulsified neat oil droplets/phospholipid-coated droplets were mixed with an equal volume of unlabeled PLIN1 AH peptide solution to achieve a 20-fold excess of unlabeled PLIN1 AH peptide over FITC-labeled peptide. Droplet images were then captured at indicated time points with a Zeiss LSM900 confocal system and analyzed with ImageJ.
4
BMAL1 Immunofluorescence Staining in Monkeys
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Immunofluorescence staining of BMAL1+/+ and BMAL1–/– cynomolgus monkey tissues was conducted as previously described (55 ,81–82 (link)). In short, the paraffin-embedded sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval after deparaffination and rehydration. Then, the sections were treated with 0.4% Triton X-100 for permeabilization and 10% donkey serum for blocking. Primary antibodies were then applied at 4°C overnight. After several washes with PBS, the sections were incubated with secondary antibodies and Hoechst 33342 (Thermo Fisher Scientific). Finally, the sections were mounted with Antifade Mounting Medium (Vector Laboratories, H-1000) after several washes with PBS. The images were obtained by a ZEISS LSM900 confocal system and the mean fluorescence intensity of H3K9me3 were measured by the ImageJ software (NIH). Antibodies used in immunofluorescence staining were listed in Supplementary Table S2 .
5
Olaparib and KPT330 Induced DNA Damage
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About 5000-20000 cells were seeded in a cell imaging dish (NEST, 801002) and exposed to olaparib and KPT330 for 48 h. After treatment, 4% paraformaldehyde was used to fix the cells for about 10 min. Cells were permeabilized in 0.5% Triton X-100 (Beyotime Institute of Biotechnology, P0096) for 15 min. After three times washing with PBS, blocking was done with 5% goat serum (Boster Biological Technology, AR0009) for 30 min at 37°C. cells were incubated at 4°C overnight with primary antibody: anti-γH2AX (Cell Signaling Technology, 9718S), anti-p-CHEK1 (Cell Signaling Technology, 2348S), anti-SQSTM1 (Cell Signaling Technology, 7695S), anti-LC3 (MBL BEIJING BIOTECH CO., LTD, M152-3). After primary antibody staining, cells were washed with PBST (PBS buffer containing 1% Tween 20) followed by PBS only washing. The goat anti-rabbit secondary antibody Alexa Fluor® 488 conjugate (Invitrogen, A32731) or goat anti-mouse secondary antibody Alexa Fluor® 555 conjugate (Invitrogen, A32727) was used as a secondary antibody (1:400 dilution). Nuclear stain (DAPI) containing ProLong Gold antifade reagent (Thermo Fisher Scientific, P36935) was added to the slides. Images were analyzed on an LSM900 confocal system (Zeiss, Jena, Germany). The number of LC3 dots was counted in 10 randomly selected cells.
6
Apoptosis Detection in Ovarian Tissues
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TUNEL staining was performed using the One Step TUNEL Apoptosis Assay Kit (Beyotime, C1088) following the manufacturer’s instruction to identify the apoptotic signals within ovarian tissues. Briefly, paraffin-embedded ovarian tissues sections (with a thickness of 5 μm) were routinely dewaxed to water. These sections were incubated with 20 μg/mL DNase-free proteinase K (dilution with 10 mmol/L Tris-HCl pH = 7.8) for 30 min at RT, followed by three rinses in PBS. Next, the sections were stained with TUNEL working solution at 37°C for 1 h. Then the slides were counterstained with Hoechst 33342 (Thermo Fisher, H3570, 1:1,000) to visualize the nucleus, and washed three times with PBS. Finally, the slides were mounted with VECTERSHIELD® anti-fading mounting medium (Neobioscience, H-1000). Image capture was performed using the Zeiss LSM900 confocal system.
7
Quantifying Senescence in Liver Tissue
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Senescence-associated β-galactosidase (SA-β-gal) staining was performed according to the instructions provided by the manufacturer (DojindoMolecular Technologies, Inc., Kumamoto, Japan). First, OCT-embedded liver tissues were cryosected at a thickness of 10 μm using a Leica CM3050S cryosectioner. Then sections were fixed in 4% paraformaldehyde for 20 min at RT and washed in PBS three times. SPiDER-βGal staining working solution was diluted to 20 μmol/L (Dojindo Molecular Technologies, SG03, 1:2,000) with McIlvaine buffer (pH 6.0) and sections were incubated with this working solution overnight at 4°C. Sections were then counterstained with Hoechst33342 (Thermo Fisher, H3570, 1:1000) at RT, washed with PBS three times, and mounted in VECTERSHIELD® Anti-Fade Mounting Medium (Neobioscience, H-1000). Image acquisition was performed using a Zeiss LSM900 confocal system, and data analysis of the percentage of SPiDER-βGal positive cells was performed using image J.
8
Immunofluorescence Analysis of Corneal IL-1β and IL-6
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Frozen corneal sections were blocked with 1% BSA and labeled with IL-1β (1:200, Abcam) and IL-6 (1:500, Abcam) primary antibodies at 4 °C overnight. The sections were incubated with corresponding Alexa Fluor-conjugated secondary antibodies (1:100, Invitrogen, USA). Photographs were taken with a Zeiss LSM 900 confocal system (Zeiss, Oberkochen, Germany). At least six images per eye at 20× magnification were collected to quantify the number of positive cells in the cornea with ImageJ software.
9
Quantification of Macrophages in Optic Nerve
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First, the ON vertical-sections were blocked using 2% bovine serum albumin (BSA) containing 0.3% triton X-100 for 1 h. ON tissue was incubated with anti-ED1 (1:50; Bio-Rad Laboratories, Inc., Berkeley, CA, USA) and anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (1:100; Abcam, Cambridge, UK) primary antibodies overnight at 4 °C. Goat anti-mouse Alexa Fluor 488 antibody (1:100, Invitrogen, Waltham, MA, USA) was used as a secondary antibody and incubated with the section for 1 hour at room temperature (RT). Fluorescent images of the ON tissue section were taken at 10x and 20× magnification using a Zeiss LSM 900 confocal system (Carl Zeiss AG, Oberkochen, Germany). The 20× magnification image of ED1+ cell was analyzed using ImageJ software for quantization of extrinsic macrophages.
10
Immunohistochemical Analysis of Optic Nerve and Retina
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The frozen sections of ONs and retina were rinsed with PBS and then blocked with 5% normal goat serum containing 1% bovine serum albumin (BSA) for 30 min. The ON sections were labeled with anti-ED1 (CD68; 1:50; Bio-Rad, Berkeley, CA, USA), anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (1:200; Abcam, Cambridge, UK), and anti-Ym1 (1:50; Abcam) primary antibodies, and the retinal sections were labeled with anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:200; Abcam) and anti-interleukin 6 (IL-6) (1:1000; Abcam) primary antibodies. Those sections were then incubated with corresponding Alexa Fluor conjugated secondary antibodies. Photographs were taken using a Zeiss LSM 900 confocal system (Carl Zeiss, Oberkochen, Germany). At least six images per eye by 20× magnification were taken for quantification the ED-1 or Ym1 staining positive cell in the optic nerve and for quantification of Iba1 and IL-6 staining in the retina. To quantify the intensity of CNPase staining in optic nerve, we opened CNPase and DAPI channel in ImageJ, the intensity of CNPase and DAPI in optic nerve were measured and the CNPase generated per DAPI-positive cells were calculated.
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