Immuno-histochemical assays were used to determine CD163 and iNOS protein expression in the bladder tissue of rats. Tissues were obtained from rats with/without HPP treatment, fixed in 10% formaldehyde for 24 h, and then embedded in paraffin. The specimens were cut into 5-μm sections and roasted at 60°C for 2 h. Antigenic retrieval was performed in citric acid buffer (pH = 6.0) followed by washing with PBS and treatment with 3% H2O2 and 5% bovine serum albumin. Afterward, the sections were incubated with primary antibodies against CD163 and iNOS (dilutions of 1:50; Cell Signaling Technology) at 4°C overnight. Subsequently, they were incubated with secondary antibody for 30 min. Detection was performed using 3,3′-diaminobenzidine (DAB) chromogen (Maixin Biotech. Co. Ltd, Fuzhou, China), and the sections were counterstained with hematoxylin, dehydrated through graded alcohols, and cleared in xylene. Finally, pictures were taken under 200× magnification under a microscope (Ti2-E; Nikon). Immunostaining was evaluated by Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, MD, USA).
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Lab products found in correlation
Ti2 e, by Nikon (2 mentions)
Gs3 u3 51s5m c camera, by Teledyne (2 mentions)
Pannoramic midi, by 3DHISTECH (2 mentions)
Cm1850, by Leica (2 mentions)
3 3 diaminobenzidine dab kit, by Wuhan Servicebio Technology (2 mentions)
Bx53 dp72 microscope, by Olympus (2 mentions)
Lsm 700, by Zeiss (2 mentions)
3 3 diaminobenzidine dab chromogen, by Maixin Group (1 mentions)
Protease inhibitor cocktail, by Promega (1 mentions)
Proteome profiler array, by R&D Systems (1 mentions)
Mouse anti d2 40, by Agilent Technologies (1 mentions)
Mouse anti e cad, by Cell Signaling Technology (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
Goat anti rabbit hrp, by Cell Signaling Technology (1 mentions)
In cell analyzer, by GE Healthcare (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Rabbit anti rat cd31 antibody, by Abcam (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Second antibody, by Maixin Group (1 mentions)
Ab40801, by Abcam (1 mentions)
29 protocols using image pro plus 6.0 image analysis software
1
Immunohistochemical Analysis of CD163 and iNOS
2
Hepatic Glycogen Content Quantification
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The hepatic glycogen content was determined by periodic acid-Schiff staining. The liver tissues were fixed as above described and embedded in paraffin, and continuously sliced into 5-µm-thick sections. The sections were oxidized with periodic acid for 10 min and stained with Schiff reagent for 15 min at room temperature. Cell nuclei were re-stained with hematoxylin for 5 min at room temperature. Then, sections were deparaffinized with xylene and sealed. Cells with red or magenta particles in the cytoplasm were defined as positive for glycogen. Tissue sections digested with amylase were used as negative controls.
For the quantification of glycogen content, six rat livers were randomly selected from each group, three sections were selected from each rat liver, and three views were observed in each section. The liver lobules with intact tissue structure were selected for observation using an light microscope (BH-2; Olympus Corporation, Tokyo, Japan; magnification, ×200). Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the integral optical density of glycogen in hepatocyte cytoplasm, and the mean value was determined as the glycogen content.
For the quantification of glycogen content, six rat livers were randomly selected from each group, three sections were selected from each rat liver, and three views were observed in each section. The liver lobules with intact tissue structure were selected for observation using an light microscope (BH-2; Olympus Corporation, Tokyo, Japan; magnification, ×200). Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculate the integral optical density of glycogen in hepatocyte cytoplasm, and the mean value was determined as the glycogen content.
3
Quantification of Growth and Inflammatory Factors in Foot Ulcers
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The growth factors and inflammatory factors were detected by ELISA and proteome profiler array. The other half of the tissue harvested from foot ulcers was cut into smaller pieces and washed in sterile PBS. Total protein was extracted, centrifuged, and resuspended in sample application buffer containing a protease inhibitor cocktail (Promega, Madison, WI, USA). The levels of growth factors and inflammatory factors in the foot ulcerative tissue were measured using the enzyme ELISA kit (Shanghai ExCell Biology, Inc., Shanghai, China) and proteome profiler array (R&D Systems, Inc., USA) in accordance with the respective product instruction manual. For growth factors, the optical density of each well was determined by spectrophotometer under a 450 nm wavelength after a substrate solution of hydrogen peroxide was added to the plate, and the concentrations of cytokines were calculated. For inflammatory factors, pixel densities on developed X-ray films were collected and analyzed using Image-Pro Plus 6.0 image analysis software (Media Cybernetics Inc, Silver Spring, MD, USA).
4
Quantifying Alzheimer's Pathology in APP/PS1 Mice
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Following the brains of APP/PS1 transgenic mice, serial 10-μm coronal sections were made with a cryostat (Leica CM 1850, Leica Microsystems AG, Wetzlar, Germany). Five mice per group were analyzed, and nine hippocampal sections from each APP/PS1 transgenic mouse were selected for immunohistochemical staining. The sections were stained with 0.05% 3, 3′-diaminobenzidine (DAB). Whole images were captured using a Pannoramic MIDI (3D Histech, Hungary) equipped with a GS3-U3-51S5M-C camera (FLIR, Canada), Lumencor SOLA (Beaverton, OR), and Semrock filters (Rochester, NY). The Aβ plaque load was quantified by the areas of Aβ plaques in the cortex and hippocampus divided by the total area of the corresponding sites. The microglia- and astrocyte-positive area were quantified as the GFAP+ or Iba1+ area divided by the total area of the hippocampus. Image-Pro Plus 6.0 image analysis software (Media Cybernetics, MD, USA) was used to analyze the images.
5
Immunohistochemical Analysis of Joint Tissues
6
Immunohistochemistry Profiling of Gastric Tumor
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Immunohistochemistry (IHC) assay was conducted as per the standard protocols. The monoclonal antibodies used for the analyses were as following: rabbit anti-CD66b (Abcam, Cambridge, UK), mouse anti-D2-40 (Dako, Carpinteria, USA), mouse anti-E-cadherin (E-cad) (Dako, Carpinteria, USA), rabbit anti-family with sequence similarity 3 member C (FAM3C) (Abcam, Cambridge, UK), rabbit anti-integrin α6 (Abcam, Cambridge, UK) and rabbit anti-CD151 (Affinity Biosciences, Cincinnati, USA) (for human tissue specimens), mouse anti-E-cad (Cell Signaling Technology, MA, USA), rabbit anti-vimentin (Vim) (Cell Signaling Technology, MA, USA) (for mouse allograft). Integrin α6 (ITGA6) and CD151 were detected using dual-color immunostaining in gastric tumor tissues. The expression intensity of E-cad or Vim was evaluated with mean density (MD) using Image-Pro Plus 6.0 image analysis software (Media Cybernetics, America), and MD means integral optical density (IOD) SUM divided by area SUM.
7
Immunohistochemical Analysis of CNN2 in HCC
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Paraffin sections were dewaxed and hydrated. Sections were retrieved by citrate buffer and incubated with 3% H2O2 at room temperature for 15 min. Goat serum was used to block nonspecific binding sites for 30 min and diluted rabbit anti-human CNN2 polyclonal antibody (1:200) was added overnight at 4 °C, followed by horseradish peroxidase-labeled anti-rabbit secondary antibody at 37 °C for 40 min. Slides were stained by diaminobenzidine (DAB) (ZLI9018, ZSGB-BIO, Beijing, China) and hematoxylin, then mounted by neutral balata. Pictures were taken by 200× magnification light microscope (MSC-B203T, Olympus, Beijing, China). Known positive HCC tissue sections were used as a positive control, and PBS was used instead of primary antibody as a blank control. Images were taken with a 200× magnification light microscope. The average optical density was calculated by image-Pro Plus 6.0 Image analysis software (version 6.0, Media Cybernetics, Rockville, MD, USA).
8
Immunohistochemical Analysis of LC3B
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Fresh kidney tissues were fixed in 4% paraformaldehyde (for 72 h at 4°C) and embedded in paraffin. Specimens were cut into 4 µm-thick sections and deparaffinized in xylene. After tissue sections were washed with PBS, they were boiled in 10 mM citrate buffer (pH 6.0) for 4 min for antigen retrieval and then blocked with 10% goat serum in PBS at room temperature for 1 h. The primary antibody (anti-LC3B; 1:400; cat. no. 4412; Cell Signaling Technology, Inc.) was added in accordance with the instructions and incubated at 4°C for 12 h. The secondary antibody (goat antirabbit HRP; 1:2,000; cat. no. BS13278; Bioworld Technology, Inc.) was added and incubated at room temperature for 10 min. DAB (100 µl) was added and counterstained for 5 min with the staining observed under a light microscope (Model CX31-P; Olympus Corporation). The intensity of positive staining, which appeared brown, was determined using Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc.). The integral optical density (IOD) was calculated to represent the intensity. IOD values increased as protein expression increased.
9
Immunocytochemical Analysis of Brain Sections
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Brain coronal sections were incubated for 1 h at room temperature with 10% goat serum then overnight at 4°C with primary antibodies to clathrin, RAB5B and NMDAR1 (Supplementary Table 1). After incubation with secondary antibodies for 50 min, immunostaining was detected with 3,3′-diaminobenzidine kit (DAB, G1211, Servicebio). Image pro plus 6.0 image analysis software (Media Cybernetics, Rockville, MD, USA) was used for data analysis. The Area and Integral Optical Density (IOD) of the positive regions were imaged and the relative protein expression levels were calculated as the mean optical density (IOD/Area).
Immuno uorescence PC12 cells were rinsed with PBS and xed with 95% ethanol or 4% paraformaldehyde. After blocked with 3% BSA for 1 h at 37°C, cells were incubated with primary antibodies (Supplementary Table 1) for 2 h at 37°C. Subsequently, cells were washed and incubated with secondary antibodies in the dark for 1 h at 37°C, followed by staining with 20μg/ mL Hoechst 33342 (Sigma-Aldrich) for 15 min. A IN Cell Analyzer high connotation cell imaging analysis system (2500HS, GE, USA) was used the observation.
Immuno uorescence PC12 cells were rinsed with PBS and xed with 95% ethanol or 4% paraformaldehyde. After blocked with 3% BSA for 1 h at 37°C, cells were incubated with primary antibodies (Supplementary Table 1) for 2 h at 37°C. Subsequently, cells were washed and incubated with secondary antibodies in the dark for 1 h at 37°C, followed by staining with 20μg/ mL Hoechst 33342 (Sigma-Aldrich) for 15 min. A IN Cell Analyzer high connotation cell imaging analysis system (2500HS, GE, USA) was used the observation.
10
Quantifying Glomerular Endothelial Cell Proliferation
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The expression of CD31 known as platelet-endothelial cell adhesion molecule-1 was detected by immunofluorescence staining in each group. Briefly, the paraffin sections were placed in an oven at 60 °C for 1 hour and then rehydrated in a graded ethanol series (100 %, 95 %, 90 %, 85 %, and 70 %). Citrate buffer was used for heat-induced antigen retrieval. Then goat serum was used as a blocking solution before the primary antibody was applied. The sections were incubated overnight in 4 °C with rabbit anti-rat CD31 antibody at a 1:150 dilution (Abcam, USA), and mouse anti-rat Ki-67 (a marker of proliferation) antibody at a 1:100 dilution (Abcam, USA).The secondary antibody, goat anti-rabbit antibody conjugated to Alexa Fluor 594 (BA1032, China) and goat anti-mouse antibody conjugated to Alexa Fluor 488 (Life, USA) were applied at 1:200 dilutions for 1 hour. Then immunofluorescence photomicrographs were obtained at 400× magnifications using a fluorescence microscope (Leica, Solms, Germany), and the intensity of the CD31-positive signal was quantified with Image pro plus 6.0 image analysis software (Media Cybernetics, USA) to represent glomerular endothelial cell proliferation.
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