For histone protein detection, we either FACS-isolated bulge and non-bulge cells or extracted chromatin from whole skin snap frozen in liquid N2. From FACS-isolated bulge and non-bulge cells, cells were first incubated in Triton Extraction Buffer (PBS containing 0.5% Triton × 100 (volume per volume), 2 mM phenylmethylsulfonyl fluoride), followed by 0.2 N HCl incubation. H3K9me3 (1:500–1: 3,000; Millipore) and H3 (1:3,000–1: 10,000; Abcam) antibodies were used for immunoblotting. For whole skin chromatin extraction, tissue samples were first lysed with Buffer A (1 M HEPES-KOH, pH7.8, 3 M KCl, 1 M MgCl2, 1 M sucrose, glycerol and protease/phosphatase inhibitors), and the pellet after spin down was incubated with Buffer B (0.5 M EDTA, 0.5 M EGTA, and protease/phosphatase inhibitors). H3K4me3 (Active motif, 1:1,000), H3K9me3 (Abcam, 1:2,000), H3K27me3 (Upstate, 1:1,000), H3 (Abcam, 1:2,000) antibodies were used for immunoblotting. ImageJ (http://ImageJ.nih.gov/ij/) and AutoQuant (MediaCybernetics) were used for normalization and quantifications. Raw western blot scanned images are included in Supplementary Fig. 5 .
Imagej
Manufactured by Media Cybernetics
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ImageJ is an open-source image processing program designed for scientific multidimensional images. It provides a wide range of tools for image analysis, processing, and visualization, making it a versatile tool for researchers and scientists working with images.
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Lab products found in correlation
Matrigel, by BD (5 mentions)
Image pro plus, by Media Cybernetics (5 mentions)
A1 plus, by Nikon (3 mentions)
Fluoview fv1200, by Olympus (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Image pro plus 5, by Media Cybernetics (2 mentions)
X omat ar film, by Kodak (2 mentions)
Tnf α, by Abcam (2 mentions)
Dm6 b, by Leica (2 mentions)
Il 1β, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Dm500, by Leica (1 mentions)
Edu staining kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole staining solution, by Beyotime (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Tunel brightred apoptosis detection kit, by Vazyme (1 mentions)
H3k9me3, by Merck Group (1 mentions)
H3k27me3, by Abcam (1 mentions)
Autoquant, by Media Cybernetics (1 mentions)
H3k9me3, by Abcam (1 mentions)
72 protocols using imagej
1
Histone Modification Detection in Skin
2
Matrigel-Based In Vitro Angiogenesis Assay
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We used 50% Matrigel (50 μl/well, BD Biosciences, CA, USA) to coat a precooled 96-well plate and it was left to polymerize at 37 °C for 30 min. Next, 3 × 104 HUVECs cells were suspended in a mixture of conditioned medium (50 μl) and endothelial cell medium (50 μl) containing 10% FBS. Tube structures were photographed after incubation for 6 h at 37 °C, and analyzed with Image J (Media Cybernetics, MD, USA) as described previously43 (link).
3
Confocal Microscopy Immunofluorescence Analysis
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4
Histological Assessment of Kidney Injury
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Paraffin-embedded one-fourth injured kidney was cut into 5-mm sections from the maximum side and subsequently deparaffinized and rehydrated. Hematoxylin and eosin (H&E) staining, Masson trichrome, and Sirius red staining were performed as previously described. Ten fields of each specimen were randomly chosen for observation at 200× magnification. For H&E staining, the severity of histological injury was evaluated randomly and independently by two renal pathologists according to the grading system described by Lin et al. Semiquantitative assessment of Sirius red and Masson trichrome was performed using ImageJ bundled with Java 1.8.0_172 (Media Cybernetics, MD, USA) to evaluate ECM deposition in the renal interstitium.
5
Quantification of Monocyte-Macrophage and Apoptosis in Aortic Lesions
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Tissue sections of the aortic sinus adjacent to the sites of maximum lesion area were dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubation with peroxoblock (Zytomed Systems GmbH, Berlin, Germany). Monocytes/macrophages were detected by using a monoclonal rat anti-mouse Mac-2-antibody (WAK-Chemie Medical GmbH, Steinbach, Germany). Anti-Mac-2 or isotype control was incubated for 1.5 hours at room temperature. The sections were then incubated with the biotinylated secondary antibodies for 30 minutes, rinsed three times with phosphate-buffered saline, and incubated for 10 minutes with streptavidin at room temperature. Acetate buffer, 3-amino-9-ethylcarbazole (AEC)-chromogen substrate (Zytomed Systems GmbH) was used for visualization. In order to detect apoptosis, “In Situ Cell Death Detection Kit TMR red” was used by the protocol of the manufacturer (Roche Diagnostics GmbH Mannheim, Germany). For quantification of staining we used computer-assisted morphometry. The results of Mac-2 staining is presented as ration extend stained area/total area of the lesion. Results of apoptosis are given as the ratio of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive areas/total lesion area (Image J; Media Cybernetics, Inc.).
6
Quantitative Image Analysis of IHC Staining
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Fiji software, an open-source image-processing package based on ImageJ (Media Cybernetics, Inc., Rockville, MD, USA), was used to measure the fractional area and integrated optical density (IOD) of the stained slides as reported in our previous study [22 (link)]. To summarize, first, the microscopic image of each tissue stained with IHC was transformed into an 8-bit grayscale. Then we created a ‘threshold’ binary image by selecting ‘Image > Adjust > Threshold’ and used the slider to adjust the threshold to match the positive areas. Finally, we called ‘Analyze > Measure’ for the results popup menu with fractional area to get the automatic percentage-stained area value. The OD was obtained using the formula: OD = log (250/Pixel value) under the calibration procedure performed on the 8-bit grayscale images. After processing the threshold of the image as described above, the IOD was measured by clicking ‘Analyze > Measure’ for the integrated density value. DAB staining intensity was determined by mean IOD (mean IOD = IOD/area). The protein levels of each section are represented as an average IOD.
7
Histological Analysis of Colonic Tissue
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Collected colon samples were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded colonic tissues were sectioned (4μm in thicknesses) and were subjected to hematoxylin and eosin (HE) and alcian blue (AB) staining by using commercial kits (Beijing Solarbio Technology Co., Ltd. Beijing, China) according to the manufacturer's instructions. The crypt height and muscular layer width in the large intestine were measured using ImageJ (Media Cybernetics, Inc., Rockville, MD, USA). Mucusproducing goblet cells were observed and counted under light microscope (Leica DM500). Six microscopic fields of every section of the testes were randomly selected.
Histological pathology was scored according to a previously established scoring system, as follows: crypt damage (0–4 scale), severity of inflammation (0–3 scale), and depth of injury (0–3 scale) [61 (link)].
Histological pathology was scored according to a previously established scoring system, as follows: crypt damage (0–4 scale), severity of inflammation (0–3 scale), and depth of injury (0–3 scale) [61 (link)].
8
EdU Staining for DNA Replication Analysis
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DNA replication activity was analyzed in cells with an EdU staining kit (Thermo Fisher Scientific, C10337). Briefly, HK-2 cells were seeded on coverslips in 12-well plates as previously described. EdU (10 μM) was added to each well for 2 h until the cells were harvested 48 h after stimulation. Cells were collected and fixed with 4% paraformaldehyde for 10 min and then were permeabilized with 0.5% Triton X-100 for 10 min at room temperature. The cells were then stained with a Click-iT® Plus reaction cocktail kit for 30 min at room temperature. Finally, images were obtained with a microscope (Nikon, Tokyo, Japan) and then were analyzed with Image J (Media Cybernetics, Silver Spring).
9
Ovarian Apoptosis Assay with TUNEL
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Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) was used to assay the apoptosis in the ovary. The whole experiment was performed using a commercial TUNEL BrightRed Apoptosis Detection Kit (A113, Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions. First, we deparaffinized, rehydrated the sections of ovary. The sections were then incubated with Proteinase K (20 μg/mL) at room temperature for 20 min. Second, the sections were incubated with the terminal deoxynucleotidyl transferase (TdT ) enzyme with BrightRed Labeling Mix at 37°C for 60 min in the dark. Finally, the sections were stained with 4′,6-diamidino-2-phenylindole staining solution (C1005, Beyotime Biotechnology, Shanghai, China) for 5 min in the dark. To ensure there was no nonspecific reaction, we performed the negative control without incubation of the TdT enzyme. The fluorescent images were acquired using a LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The bright of apoptotic cells (red color) was measured using the Image-J (Media Cybernetics, Bethesda, MD).
10
Retinal Astrocyte Reactivity Analysis
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