Five μm thin paraffin sections of ileal ex vivo biopsies were used for in situ immunohistochemical analyses as described previously [15 (link), 18 (link), 19 (link)]. Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Boston, MA, USA, 1:200), CD3 (Polyclonal rabbit anti human, DAKO, Denmark; 1:10), FOXP3 (FJK-165, eBioscience, Germany; 1:100), B220 (eBioscience; 1:200) and F4/80 (biot. Clone BM 8 rat anti-mouse, Life Technologies, USA; 1:100) were used to assess apoptotic cells, T lymphocytes, regulatory T cells (Treg), B lymphocytes and macrophages / monocytes, respectively. The average number of positively stained cells within at least six high power fields (HPF, 0.287 mm2; 400 x magnification) were determined by an independent and blinded investigator.
Polyclonal rabbit anti human
Manufactured by Agilent Technologies
Sourced in United States
Polyclonal rabbit anti-human is a laboratory reagent used in various immunoassay and research applications. It is derived from rabbit serum and contains a mixture of antibodies that recognize multiple epitopes on human proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mantra system, by PerkinElmer (2 mentions)
Inform automated image analyses software, by PerkinElmer (2 mentions)
Asp175, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Polyclonal rabbit anti mouse, by Agilent Technologies (1 mentions)
Polyclonal swine anti rabbit igg, by Agilent Technologies (1 mentions)
Dako omnis autostainer, by Agilent Technologies (1 mentions)
5 protocols using polyclonal rabbit anti human
1
Immunohistochemical Analysis of Ileal Biopsies
2
Antibody Staining for Viral Detection
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Antibodies used for immunofluorescence (IF) and/or flow cytometry were:- Monoclonal anti-measles (IgG1, Oxford Biotechnology, dilution 1:100) and secondary antibodies Alexa Fluor 488 goat anti-mouse (Molecular Probes, dilution 1:500) was used for MV. Palivizumab 100 mg/ml was used as primary antibody for RSV (1:1000 dilution) and polyclonal rabbit anti-human (Dako, dilution 1:200) as secondary antibody. Rabbit polyclonals to ASIC3, TRPV1 and TRPA1 (Abcam. dilution 1:50) or non-immune rabbit serum (as control, dilution 1:50) were used with secondary antibody goat polyclonal to rabbit IgG (Chromeo™ 488, dilution 1/500). Detailed methods for both IF and flow cytometry are given in the supplementary information S1 File .
3
Immunohistochemical Characterization of Endothelial Cells
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HPAECs were seeded (5 × 104−1 × 105 cells) onto sterile 12 mm glass coverslips in a 24-well plate. At confluency they were fixed in 100% cold methanol (30 min) and stored under sterile PBS for use in immunohistochemistry. They were stained for endothelium-specific markers: mouse monoclonal anti-human CD31 (CD31; Dako, cat no. M0823, 1 in 50), VEGF receptor 2 (VEGFR2; anti-VEGF R2/KDR fluorescein conjugated mouse IgG; R&D Systems, cat no. FAB357F, 1 in 10), von Willebrand factor (polyclonal rabbit anti-human, Dako, cat no. A0082, stock concentration 3.1 mg ml−1, 1 in 250), and Ulex europaeus agglutinin 1 lectin (direct fluorescent conjugate; Vector Laboratories, 1 in 25). Smooth muscle α-actin was used as a negative control (mouse monoclonal anti-human; Dako, cat no. M085, 1 in 100). Vectashield mounting medium was used with 4’,6-diamidino-2-phenylindole (DAPI). Secondary antibodies, fluorescently labelled with fluorescein isothiocyanate, were used accordingly: (i) polyclonal rabbit anti-mouse (Dako, cat no. F0232, 1 in 50); and (ii) polyclonal swine anti-rabbit IgG (Dako, cat no. F0205, 1 in 50).
4
Automated Quantification of CD3+ T-cells
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IHC staining for CD3 was conducted using the Dako Omnis autostainer. Briefly, tissue sections 4 µm thick were boiled and then subjected to dewaxing, rehydrating, and antigen retrieval, followed by incubation with an anti-CD3 primary antibody (Dako Omnis, polyclonal rabbit anti-human, Santa Clara, CA, USA). For signal visualization, the EnVision FLEX+ High pH (Link) system was used, following the manufacturer’s instructions. The sections were manually mounted in neutral resin for observation under a light microscope. Whole-slide images were acquired using the light microscope on the Mantra System (PerkinElmer, Waltham, Massachusetts, USA). Images were used to quantify the CD3 signal, and the T-cell levels were calculated using the inForm automated image analyses software (PerkinElmer, Waltham, Massachusetts, USA). Briefly, CD3+ cells were recognized by machine-learning-based classification according to CD3 staining signal and the percentage was calculated as the number of CD3+ cells divided by the total number of nucleated cells in all view fields.
5
Automated Quantification of CD3+ T Cells
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IHC staining for CD3 was conducted using the Dako Omnis autostainer. Briefly, sections 4 μm thick were boiled and then subjected to dewaxing, rehydrating, and antigen retrieval, followed by incubation with an anti‐CD3 primary antibody (Dako Omnis, polyclonal rabbit anti‐human, Santa Clara, CA, USA). For signal visualization, the EnVision FLEX+ High pH (Link) system was used, following the manufacturer's instructions. The sections were manually mounted in neutral resin for observation under a light microscope. Whole‐slide images were acquired using the light microscope on the Mantra System (PerkinElmer, Waltham, Massachusetts, US). Images were used to quantify the CD3 signal, and the T cell levels was calculated using the inForm automated image analyses software (PerkinElmer, Waltham, Massachusetts, USA). Briefly, CD3+ cells and other cells were recognized by machine‐learning‐based classification according to CD3 staining signal and the percentage was calculated.
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