Frozen brain sections or cells were fixed with 4% PFA for 20 min at room temperature and were permeabilized with 1% Triton X‐100 (Solarbio, Beijing, China) solution for 15 min. Next, 5% BSA in PBS with 0.1% triton was used to block non‐specific binding sites at room temperature for 1 h. The slides were incubated with the following corresponding primary antibodies at 4°C overnight: mouse anti‐dsDNA (1:100, Santa Cruz Biotechnology); rabbit anti‐53BP1 (1:5,000, Novus Biologicals); rabbit anti‐Iba1 (1:500, Wako Pure Chemical Industries, Ltd., Japan); rabbit anti‐NeuN (1:500, Abcam); goat anti‐GFAP (1:500, Abcam); rat anti‐Ly6G (1:100, BioLegend); mouse anti‐cGAS (1:100, Santa Cruz Biotechnology); mouse anti‐GSDMD (1:100, Santa Cruz Biotechnology); mouse anti‐caspase‐1 (1:100, Adipogen); and goat anti‐IL‐1β (1:100, R&D Systems). The following day, the sections were washed with cold PBS; the immunoreactions were visualized using fluorescent secondary antibodies. Nuclei were costained with Fluoroshield Mounting Medium containing DAPI (104139, Abcam). All the slides were visualized and photo‐documented using a confocal microscope (Olympus, Heidelberg, Germany) or a Nikon Coolscope digital microscope (Nikon, Tokyo, Japan), and quantified by ImageJ software (NIH).
Coolscope digital microscope
Manufactured by Nikon
Sourced in Japan
The Coolscope digital microscope by Nikon is a compact and versatile microscope designed for laboratory use. It features a high-resolution digital camera and a range of optical configurations to accommodate various sample types and magnification requirements. The Coolscope is capable of capturing detailed images and transmitting them to a connected computer for further analysis and documentation.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse caspase 1, by Adipogen (2 mentions)
Mouse anti gsdmd, by Santa Cruz Biotechnology (2 mentions)
Mouse anti cgas, by Santa Cruz Biotechnology (2 mentions)
Goat anti il 1β, by R&D Systems (2 mentions)
Rat anti ly6g, by BioLegend (1 mentions)
Fluoroshield mounting medium containing dapi, by Abcam (1 mentions)
Rabbit anti 53bp1, by Novus Biologicals (1 mentions)
Goat anti gfap, by Abcam (1 mentions)
Rabbit anti neun, by Abcam (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Facsaria 3, by BD (1 mentions)
Tunel kit, by Roche (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (1 mentions)
Anti erβ, by Abcam (1 mentions)
Prism 9, by GraphPad (1 mentions)
Prism 7, by GraphPad (1 mentions)
7 protocols using coolscope digital microscope
1
Immunofluorescence Imaging of Cellular Markers
2
Quantifying Cell Apoptosis in MCAO Model
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Three days after MCAO, flow cytometry was performed to evaluate cell apoptosis in the brain tissues with indicated groups using the apoptosis assay datasheet (Solarbio, Beijing, China) with a FACS Aria III (BD Bioscience, San Jose, CA, USA) according to the manufacturers’ instructions. Data were analyzed with FlowJo software (Version 7.6.1, FlowJo, LLC). Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining was used to access the extent of cell death using a TUNEL kit (Roche, USA). Slides were observed and photographed using a Nikon Coolscope digital microscope (Nikon, Tokyo, Japan). The TUNEL‐positive cells showed green nuclear staining, and all of the cells with blue nuclear DAPI staining were counted within five randomly chosen fields. The index of apoptosis was expressed as the ratio of positively stained apoptotic cells to nuclei × 100%.
3
Immunohistochemical Analysis of Inflammatory Markers
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Immunohistochemical analysis was performed as previously described (McKenzie et al, 2018 ). Frozen sections (8 μm) were incubated with 0.3% hydrogen peroxide for 20 min to inactivate endogenous peroxidases. After washing in PBS, brain sections were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Thereafter, brain sections were incubated 4°C overnight with the following primary antibodies: mouse anti‐cGAS (1:100, Santa Cruz Biotechnology); mouse anti‐caspase‐1 (1:100, Adipogen); goat anti‐IL‐1β (1:100, R&D Systems); and mouse anti‐GSDMD (1:100, Santa Cruz Biotechnology). Slides were then washed in PBS and incubated with suitable biotinylated secondary antibodies (1: 500, Vector Laboratories) for 2 h at room temperature. The sections were followed by incubation with avidin–biotin–peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories). The immunoreactivity was visualized with 3,3′‐diaminobenzidine (DAB). A set of sections was also stained in a similar way but without the primary antibody and served as the negative control. Sections were then mounted in neutral balsam. All slides were imaged using a Nikon Coolscope digital microscope (Nikon, Tokyo, Japan).
4
Immunohistochemical Analysis of Duodenal ER-β
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Formalin fixation and paraffin embedding duodenal biopsy sections (4 µm) were mounted on slides, deparaffinized with xylene and rehydrated through a series of graded alcohols. Sections were incubated overnight at 4°C with anti-ER-β (Abcam, Cambridge, UK) at a 1:400 dilution in phosphate-buffered saline/bovine serum albumin-1.5%, then incubated with secondary anti-rabbit antibody for 15 min at room temperature and then in 3,3-diaminobenzidine tetrahydrochloride (DAKO) for 1 min. Sections were counterstained with hematoxylin.
The images were acquired by a Coolscope Digital Microscope (Nikon Instruments Europe, Amsterdam, Netherlands) and analyzed using the Image J program with a plugin named IHC profiler (33) . This tool has been developed for clinical histopathological sample analysis and adopts the spectral deconvolution method of diaminobenzidene (DAB)/hematoxylin color spectra using optimized optical density vectors for proper separation of the DAB color spectra. DAB-stained images undergo pixel-by-pixel analysis generating a score according to the algorithm that has been thoroughly tested (33) . DAB scores were obtained analyzing nine different images for each condition.
The images were acquired by a Coolscope Digital Microscope (Nikon Instruments Europe, Amsterdam, Netherlands) and analyzed using the Image J program with a plugin named IHC profiler (33) . This tool has been developed for clinical histopathological sample analysis and adopts the spectral deconvolution method of diaminobenzidene (DAB)/hematoxylin color spectra using optimized optical density vectors for proper separation of the DAB color spectra. DAB-stained images undergo pixel-by-pixel analysis generating a score according to the algorithm that has been thoroughly tested (33) . DAB scores were obtained analyzing nine different images for each condition.
5
Cell Quantification via Microscopy
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Semi‐quantitative analysis was performed; 15 (CD3) or 21 (CCR5) images were acquired per section at a magnification of 20× on a Nikon Coolscope digital microscope and manually counted. Counts were recorded as total positive cells per 15 or 20 images for each animal or human subject. Statistical analysis between groups was performed in GraphPad Prism 9.0.
6
Quantifying Brain Immunohistochemistry in ImageJ
7
Quantitative Analysis of Lymphoid Nodule Composition
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Sections stained with CD3 and CD79a were entirely scanned at low magnification (40x) with a Coolscope digital microscope (Nikon, Minato, Japan). The obtained images were processed with an image analysis software (ImageJ, National Institutes of Health, USA) to calculate the amount of CD3 and CD79apositive areas, expressed as percentage of the total area of the nodule in the section that was positively immunolabeled.
The sections with CD79a/Ki-67 double labelling were examined at medium magnification (100x) in order to select the areas of highest proliferative activity. Within these areas, 10 fields at 400x were photographed; 5 photographs each were taken in the CD79a-positive and CD79a-negative areas. In every image, the number of Ki-67 positive and negative nuclei was assessed with a digital cell counter (ImageJ). The Ki-67 index of CD79a-positive and -negative cells was calculated as the mean percentage of Ki-67-positive nuclei in the 5 photographed fields.
The sections with CD79a/Ki-67 double labelling were examined at medium magnification (100x) in order to select the areas of highest proliferative activity. Within these areas, 10 fields at 400x were photographed; 5 photographs each were taken in the CD79a-positive and CD79a-negative areas. In every image, the number of Ki-67 positive and negative nuclei was assessed with a digital cell counter (ImageJ). The Ki-67 index of CD79a-positive and -negative cells was calculated as the mean percentage of Ki-67-positive nuclei in the 5 photographed fields.
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