Pig's head was severed through the atlanto-axial joint. The scalp was separated from the skull and the cranium was opened with frontal and temporal incisions. The dura was cut along the lateral margins and reflected up to the midline. Finally, the whole brain was removed by severing the cranial nerve roots and the spinal cord above the first cervical vertebrae. The brain was fixed in 10% formaldehyde for posterior histological and immunochemistry analyses. First, we scored presence of cerebral petechial hemorrhages upon gross examination using the following scale: 1 = low, 2 = medium, 3 = high. Secondly, to assess histological patterns, we prepared 4 μm sections of the dentate gyrus, which were embedded in paraffin and stained with hematoxylin and eosin (H&E). Per each section, we assessed apoptosis of five areas through light microscopy (DM250, Leica, Wetzlar, Germany): cleaved caspase-3 assay (Asp175, Cell Signaling, Asp 175, Cell Signaling Technology, Inc. Danvers, MA), and TUNEL assay (In Situ Cell Death Detection Kit, Roche Diagnostics, Indianapolis, Ind). ImageJ software (National Institutes of Health; Bethesda, Mass) was used to quantify eosinophilic neuronal necrosis in H&E sections and immune-positive cells in caspase-3 and TUNEL assays (20) .
Asp175
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
Asp175 is a laboratory reagent used to detect the presence of cleaved caspase-3, a protein that is a marker for apoptosis, or programmed cell death. It is a primary antibody that specifically binds to the cleaved form of caspase-3, allowing for the identification and quantification of apoptotic cells in research samples.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 clone fjk 165, by Thermo Fisher Scientific (22 mentions)
Fjk 16, by Thermo Fisher Scientific (21 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (6 mentions)
F4 80, by Thermo Fisher Scientific (4 mentions)
Cd3 no n1580, by Agilent Technologies (4 mentions)
Ki67 tec3, by Agilent Technologies (4 mentions)
N1580, by Agilent Technologies (3 mentions)
No 14 0452 81, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Ab16667, by Abcam (2 mentions)
Anti ki67, by Thermo Fisher Scientific (2 mentions)
Ecl plus kit, by Cytiva (2 mentions)
H 125, by Santa Cruz Biotechnology (2 mentions)
Ep436y, by Abcam (2 mentions)
Ab103580, by Abcam (2 mentions)
Ab11042, by Abcam (2 mentions)
Anti tap63, by BioLegend (2 mentions)
Img 246, by Novus Biologicals (2 mentions)
Img 259a, by Novus Biologicals (2 mentions)
134 protocols using asp175
1
Histological Analysis of Pig Brain Trauma
2
Quantification of Colonic Immune Cells
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5-μm-sections of colonic tissue samples were fixed in 5% formalin and embedded in paraffin as previously described (Heimesaat et al., 2018 (link); Foote et al., 2023 (link)). Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, United States, 1:200), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), CD3 (no. N1580, Dako, 1:10), and FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100) were used to count apoptotic epithelial cells, neutrophils, T lymphocytes, and regulatory T cells under light microscopy. The mean number of detected cells in each blinded sample was determined within at least six high power fields (HPF, 0.287 mm2, 400 × magnification).
3
Quantitative In Situ Immunohistochemical Analysis
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Quantitative in situ immunohistochemical analyses were performed in colonic ex vivo biopsies following immediate fixation in 5% formalin and embedding in paraffin as recently reported [44 (link),45 (link)]. In brief, to detect apoptotic epithelial cells, macrophages/monocytes, T lymphocytes, regulatory T cells, and B lymphocytes, colonic paraffin sections (5 µm) were stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (no. N1580, Dako, 1:10), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100), and B220 (no. 14-0452-81, eBioscience; 1:200), respectively. Positively stained cells were quantitated by a blinded independent investigator applying light microscopy. The average number of respective positively stained cells in each sample was determined within at least six high power fields (HPF, 0.287 mm2, 400× magnification).
4
Immunohistochemical Analysis of Colonic Sections
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In situ immunohistochemical analysis of colonic paraffin sections was performed as described previously [13 (link), 17 (link), 18 (link), 23 (link), 24 (link)]. In brief, primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:200), CD3 (#N1580, Dako, Denmark, dilution 1:10), FOXP3 (FJK-16s, eBioscience, 1:100), B220 (eBioscience, 1:200), and F4/80 (# 14–4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50) were used [13 (link)]. For each animal, the average number of positively stained cells within at least six high power fields (HPF, 400 x magnification) were determined microscopically by a double-blinded investigator [13 (link)].
5
Quantitative Immunohistochemical Analysis of Intestinal Inflammation
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In situ immunohistochemical analysis of ileal and colonic paraffin sections was performed as previously described (25 (link)–28 (link)). Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Glostrup, Denmark, 1:100), CD3 (#N1580, Dako, 1:10), Foxp3 (FJK-16s, eBioscience, San Diego, CA, USA, 1:100), B220 (eBioscience, 1:200), and F4/80 (# 14-4801, clone BM8, eBioscience, 1:50) were used. For each animal, the average number of positively stained cells within at least six high power fields (HPF, 400× magnification) was determined microscopically by an independent blinded investigator.
6
Immunofluorescence Analysis of Neuronal Markers
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For immunofluorescence, DRG sections were incubated at 4°C overnight with a mixture of mouse anti-NeuN antibody (which targets NeuN, a neuron marker; MAB377; 1:200; Merck Millipore) and either mouse anti-p53 antibody (1C12; 1:200; Cell Signaling Technology) or rabbit anti-cleaved-caspase-3 antibody (ASP175; 1:200; Cell Signaling Technology). After washing in phosphate-buffered saline (PBS), the sections were incubated with a mixture of fluorescein-conjugated donkey anti-rabbit secondary antibody (A0453; 1:500; Beyotime) and fluorescein-conjugated goat anti-mouse secondary antibody (A0428; 1:500; Beyotime) for 1 h at room temperature away from light. The sections were washed again and dried. Finally, coverslips were placed on the slides with Gel Mount aqueous mounting medium. Images were captured using fluorescence microscopy (Olympus).
7
Quantitative In Situ Immunohistochemistry of Colonic Cells
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Quantitative in situ immunohistochemical analyses were performed in colonic ex vivo biopsies following immediate fixation in 5% formalin and embedding in paraffin as reported previously [21 (link), 25 (link)]. In brief, to detect apoptotic epithelial cells, macrophages and monocytes, neutrophils, T lymphocytes, regulatory T cells, and B lymphocytes, colonic paraffin sections (5 µm) were stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA; 1:200), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA; 1:50), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), CD3 (no. N1580, Dako, Glostrup, Denmark; 1:10), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, San Diego, CA, USA; 1:100) and B220 (no. 14-0452-81, eBioscience, San Diego, CA, USA; 1:200), respectively. Positively stained cells were quantitated by a blinded independent investigator applying light microscopy. The average number of respective positively stained cells in each sample was determined within at least six high power fields (HPF, 0.287 mm2; 400 × magnification).
8
Immunohistochemical Analysis of Colonic Biopsies
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In situ immunohistochemical analyses were performed in colonic ex vivo biopsies that had been immediately fixed in 5% formalin and embedded in paraffin as described earlier [44 (link)–46 (link)]. Paraffin sections (5 μm) of ex vivo biopsies from colon, liver and kidneys were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200) for detection of apoptotic epithelial cells; against Ki67 (TEC3, Dako, Denmark, 1:100) for detection of proliferating epithelial cells; against F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50) for detection of macrophages/monocytes; against CD3 (#N1580, Dako, 1:10) for detection of T lymphocytes; and against B220 (No. 14-0452-81, eBioscience; 1:200) for detection of B lymphocytes. Secondary antibodies were used for detection as previously described [31 (link), 47 (link)]. Positively stained cells were examined by light microscopy (magnification 100× and 400×), and for each mouse the average number of respective positively stained cells was determined within at least six high power fields (HPF, 0.287 mm2, 400× magnification) by an independent investigator using blinded samples.
9
Comprehensive Immunohistochemical Analysis of Colonic Biopsies
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Five µm thin paraffin sections of colonic ex vivo biopsies were subjected to in situ immunohistochemical analysis as described previously [27 (link)–29 (link)]. In brief, primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Boston, MA, USA, 1:200), Ki67 (TEC3; Dako, Glostrup, Denmark; 1:100), CD3 (#N1580; Dako; 1:10), FOXP3 (FJK-16 s; eBioscience, San Diego, CA, USA; 1:100), B220 (eBioscience; 1:200) and myeloperoxidase (MPO-7, # A0398; Dako; 1:500) were used to assess apoptotic cells, proliferating/regenerating cells, T lymphocytes, regulatory T cells (Treg), B lymphocytes and neutrophils, respectively. The average number of positively stained cells within at least six high power fields (HPF, 0.287 mm2; 400× magnification) were determined by an independent and blinded investigator.
10
Immunohistochemical Analysis of Colonic Biopsies
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In situ immunohistochemical analyses were carried out in colonic ex vivo biopsies immediately fixed in 5% formalin and embedded in paraffin, as previously described [41 (link),48 (link),49 (link),50 (link)]. For detection of apoptotic and proliferating epitshelial cells, macrophages/monocytes, T lymphocytes, regulatory T cells, and B lymphocytes, 5 μm thin colonic paraffin sections were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Glostrup, Denmark, 1:100), F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (#N1580, Dako, 1:10), FOXP3 (clone FJK-165, #14-5773, eBioscience, 1:100), and B220 (No. 14-0452-81, eBioscience; 1:200), respectively. Positively stained cells were enumerated using light microscopy, and the average number within at least 6 high-power fields (HPF, 0.287 mm2, 400× magnification) was recorded by an unbiased investigator.
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