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).
Foxp3 clone fjk 165
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
FOXP3 (clone FJK-165) is a monoclonal antibody that recognizes the Forkhead box P3 (FOXP3) transcription factor. FOXP3 is a key regulator of the development and function of regulatory T cells (Tregs).
Automatically generated - may contain errors
Lab products found in correlation
Asp175, by Cell Signaling Technology (22 mentions)
Cd3 no n1580, by Agilent Technologies (3 mentions)
Cd3 n1580, by Agilent Technologies (2 mentions)
No 14 0452 81, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
24 protocols using foxp3 clone fjk 165
1
Quantification of Colonic Immune Cells
2
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).
3
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).
4
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.
5
Quantitative Immunohistochemical Analysis of Colonic Biopsies
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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 [19 (link), 23 (link)]. In brief, in order to detect apoptotic epithelial cells, proliferating epithelial 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 (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).
6
Quantifying Immune Cells in Intestinal Tissue
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In situ immunohistochemical analyses were performed in 5 μm thin large intestinal paraffin sections for quantification of apoptotic epithelial cells, macrophages and monocytes, T lymphocytes, regulatory T cells and B lymphocytes by using primary antibodies directed 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, Glostrup, Denmark; 1:10), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100) and B220 (no. 14-0452-81, eBioscience; 1:200), respectively [37 (link)]. The average number of positively stained cells in each section was determined within at least six high-power fields (HPF, 0.287 mm2, 400× magnification, blinded investigator).
7
Immunohistochemical Analysis of Colonic Samples
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Immunohistochemical stainings were performed in ex vivo colonic samples that had been fixed in 5% formalin and embedded in paraffin, as previously reported [36 (link)]. In brief, to detect apoptotic cells, neutrophils and regulatory T cells, as well as T and B lymphocytes, the paraffin sections were stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), FOXP3 (clone FJK-165, No. 14-5773, eBioscience, San Diego, CA, USA, 1:100), CD3 (No. N1580, Dako, Glostrup, Denmark, 1:10), and B220 (No. 14-0452-81, eBioscience, San Diego, CA, USA, 1:200), respectively. An independent investigator determined the mean numbers of positive cells in 6 high-power fields (HPF, 0.287 mm2; 400× magnification).
8
Quantitative Immunohistochemical Analysis of Colonic Cells
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Quantitative in situ immunohistochemical analyses were performed in immediately fixed 5-µm-thin colonic paraffin sections as recently reported [60 (link),61 (link)]. In brief, in order to detect apoptotic epithelial cells, macrophages and 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), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA; 1:50), 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. An independent investigator enumerated positively stained cells by applying light microscopy and assessed the mean number of positive cells in each sample within six high power fields (HPF, 0.287 mm2, 400× magnification).
9
Quantification of Distinct Immune Cell Populations in Colonic Tissue
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The distinct immune cell population was quantified in colonic paraffin sections by applying in situ immunohistochemistry, as described earlier [42 (link),43 (link),44 (link),45 (link)]. In brief, apoptotic epithelial cells, macrophages/monocytes, T lymphocytes, regulatory T cells (Tregs), and B lymphocytes were detected in 5 μm colonic paraffin sections stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), 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 then 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 a blinded investigator.
10
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 [28 (link), 31–33 (link)]. In brief, in order to detect apoptotic epithelial cells, proliferating epithelial cells, T lymphocytes, regulatory T cells (Tregs), and B lymphocytes, 5 μm thin paraffin sections of ex vivo biopsies were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Denmark, 1:100), 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 then examined by light microscopy (magnification 100 x and 400 x), 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 x magnification) by a blinded independent investigator.
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