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.
N1580
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The N1580 is a laboratory equipment product from Agilent Technologies. It is designed to perform core functions, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Asp175, by Cell Signaling Technology (3 mentions)
No 14 0452 81, by Thermo Fisher Scientific (2 mentions)
Fjk 16, by Thermo Fisher Scientific (2 mentions)
F4 80 clone bm8, by Thermo Fisher Scientific (1 mentions)
Ncl l cd4 368, by Leica (1 mentions)
M0876, by Agilent Technologies (1 mentions)
Ultravision protein block, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi h 1200, by Vector Laboratories (1 mentions)
Bond 3, by Leica (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
9 protocols using n1580
1
Immunohistochemical Analysis of Colonic Biopsies
2
Colonic Immunohistochemical Analysis
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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 (Alutis et al., 2015a (link),b (link); Heimesaat et al., 2018 (link)). In brief, in order to detect apoptotic epithelial cells, proliferating epithelial cells, macrophages/monocytes, T lymphocytes and regulatory T cells, colonic paraffin sections (5 μm) were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Denmark, 1:100), F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (#N1580, Dako, 1:10), and FOXP3 (FJK-16s, eBioscience, 1:100), 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 × magnification) by a blinded independent investigator.
3
Quantitative Immunohistochemical Analysis of Ileum
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In situ immunohistochemical analysis of ileum paraffin sections were performed as described previously [28] (link). Primary antibodies against CD3 (#N1580, Dako, Denmark, dilution 1∶10), FOXP-3 (FJK-16s, eBioscience, 1∶100), myeloperoxidase-7 (MPO-7, # A0398, Dako, 1∶500), 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 independent high power fields (HPF, 0.287 mm2; 400× magnification) was determined microscopically by two independent double-blinded investigators.
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In situ immunohistochemical analysis of ileum paraffin sections were performed as described previously [28] (link). Primary antibodies against CD3 (#N1580, Dako, Denmark, dilution 1∶10), FOXP-3 (FJK-16s, eBioscience, 1∶100), myeloperoxidase-7 (MPO-7, # A0398, Dako, 1∶500), 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 independent high power fields (HPF, 0.287 mm2; 400× magnification) was determined microscopically by two independent double-blinded investigators.
4
Immunohistochemical and Histological Analysis of Spinal Cord
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Spines were fixed with 4% formaldehyde for 48 h, decalcified with EDTA for 1 week, followed by paraffin embedding and sectioning. Spine sections were first deparaffinized, then rehydrated with distilled water, and were subsequently incubated with primary antibodies specific against CD3 (N1580, Dako, Glostrup), F4/80 (clone BM8, eBioscience), or Iba-1 (cat number 019-19741, Wako) for 30 min, followed by staining with biotinylated donkey anti-rat or donkey anti-rabbit secondary antibodies (Dianova). A streptavidin-AP kit was used for detection. For Fast Blue staining, spine sections were stained with Luxol Fast Blue solution at 58 °C overnight. After washing with 95% alcohol and distilled water, sections were stained with a lithium carbonate solution followed by an additional round of washing with 70% alcohol and distilled water.
5
Immunohistochemical Analysis of Endomyocardial Biopsies
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Buffered formalin-fixed and paraffin-embedded (FFPE) tissues obtained from endomyocardial biopsies were subjected to immunohistochemistry staining. FFPE tissues were cut into 4-μm-thick sections and stained with hematoxylin, eosin, and Masson’s trichrome stains for morphological observation. The immunohistochemical study was performed using primary antibodies against CD3 (#N1580, Dako, Glostrup, Denmark) for T lymphocytes, CD4 (NCL-L-CD4-368, Leica Biosystems, Wetzlar, Germany) for helper T lymphocytes, CD8 (NCL-CD8-4B11, Leica Biosystems) for cytotoxic T lymphocytes, and CD68 (M0876, Dako) for macrophages. In addition, a commercially available tenascin-C antibody (4F10TT, #10337, Immuno-Biological Laboratory Co., Ltd., Gunma, Japan) was also used. We used BOND-III automated immunohistochemical staining system (Leica Microsystems K.K., Tokyo, Japan). After immunostaining, CD3-, CD8-, and CD68-positive cells were counted by two pathologists in a high-power field in the most intensively lymphocyte-infiltrated area. Each sample was classified using the grading system reported by Palaskas et al. [13 (link)].
6
Characterization of FoxP3+ Cells in EC
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In order to characterize the FoxP3-expressing cells, double immunofluorescence staining was performed. 5 specimens of EC-patients were incubated with a mouse anti-FoxP3 antibody (Dilution 1:50; 236AIE7, Thermo Fisher Scientific, Waltham, MA, USA) and a rabbit anti-CD3 antibody (ready to use; N1580, DAKO, Agilent technologies, Santa Clara, CA, USA) or a rabbit anti-CCR4 antibody (Dilution 1:50; HPA031613, Atlas Antibodies, Bromma, SWE) after blocking with Ultra-Vision-Proteinblock (Thermo Fisher Scientific, Waltham, MA, USA). Goat-anti-mouse-Alexa-Fluor488- and Goat-anti-rabbit-Cy-3-conjugated antibodies (both Dianova, Hamburg, Germany) were used as secondary antibodies. Samples were fixed with Vectashield® H1200 mounting medium with DAPI (VectorLab, Burlingame, CA, USA) and analysed using an Axiophot fluorescent photomicroscope (Zeiss, Oberkochen, Germany) and AxioVision 4.8.1 Software.
7
Immunohistochemical Staining of Colon Explants
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Immunohistochemical stainings were done in large intestinal ex vivo explants, following immediate fixation of the tissues in 5% formalin and embedding in paraffin as recently reported [63 (link),64 (link),65 (link),66 (link)]. For enumerating apoptotic epithelial cells, 5 μm thin colonic paraffin sections were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA; 1:200); for proliferating epithelial cells against Ki67 (TEC3, Dako, Glostrup, Denmark; 1:100); for macrophages/monocytes against F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA; 1:50); for T lymphocytes against CD3 (#N1580, Dako; 1:10); for regulatory T cells (Tregs) against FOXP3 (clone FJK-165, #14-5773, eBioscience; 1:100); and for B lymphocytes against B220 (No. 14-0452-81, eBioscience; 1:200). Positively stained cells were enumerated by an 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
Histopathological Analysis of Colon Tissue
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Histopathological changes were determined in GI tissue samples immediately fixed in 5% formalin and embedded in paraffin. Sections (5 µm) were stained with H&E and examined by light microscopy (100 x and 400 x magnification). In situ immunohistochemical analysis of paraffin sections taken from the colon was performed as described previously [38] (link). A primary antibody against CD3 (#N1580, Dako, Denmark, dilution 1∶10) was used to visualize T lymphocytes in the colonic mucosa in situ (200 x magnification).
9
Immunohistochemical Profiling of EMB Tissue
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Serial unstained glass slides with 4-μm thick sections from preserved buffered formalin-fixed paraffin-embedded EMB tissue from each patient were stained with hematoxylineosin (HE) and Masson's trichrome for conventional histology and fibrosis assessment, and by immunohistochemistry to detect inflammatory cells using an autostainer Leica Bond-III (Leica Biosystems, Wetzlar, Germany) in the National Cardiovascular Center, as previously described. 26, 27 Primary antibodies were anti-CD3 (N1580, Dako, Glostrup, Denmark, dilution 1 : 10) for T-lymphocytes, and anti-CD68 (PG-M1, N0876, Dako, dilution 1 : 1,000) for macrophages. Antibody detection and counter-staining with hematoxylin were performed using a Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems). To exclude the possibility of false-positives from the secondary antibody or non-specific immunoglobulin G (IgG) binding, mouse IgG1 antibody (X0931; Dako) was used as negative control.
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