Paraffin sections of formalin fixed kidneys were cut (1~2 μm) and incubated with anti-B220 (clone RA3-6B2, eBioscience Hatfield UK) or subjected to a heat-induced epitope retrieval step prior to incubation with primary antibody for CD3 (clone M-20, Santa Cruz Biotechnology, Dallas, Texas, USA), CD4 (clone 4SM95, eBioscience Hatfield UK) or MPO (polyclonal rabbit, Dako Denmark, Glostrup Denmark) followed by incubation with biotinylated rabbit anti-rat, rabbit anti-goat or donkey anti-rabbit (Dianova, Hamburg, Germany). For detection, sections were incubated with alkaline phosphatase (AP) labelled streptavidin (Dako Denmark, Glostrup Denmark). AP was visualized using Fast Red as chromogen (Dako Denmark, Glostrup Denmark). For detection of regulatory T cells, sections stained for CD4 were subjected to protein inactivation step prior to incubation with anti-Foxp3 (clone FJK-16s, eBioscience Hatfield UK). For detection, EnVision+ System- HRP Labelled Polymer Anti-Rabbit (Dako Denmark, Glostrup Denmark) was used. HRP was visualized with diaminobenzidine (Dako Denmark, Glostrup Denmark) as chromogen. Nuclei were counterstained with hematoxylin and slides coverslipped with glycerol gelatine (Merck, Kenilworth, NJ, USA). Negative controls were performed by omitting the primary antibody.
Fast red
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Fast Red is a fluorescent dye used in analytical chemistry and life science applications. It is designed for rapid and sensitive detection of target analytes in various assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti foxp3 clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
Glycerol gelatine, by Merck Group (1 mentions)
Anti b220 clone ra3 6b2, by Thermo Fisher Scientific (1 mentions)
Cd3 clone m 20, by Santa Cruz Biotechnology (1 mentions)
Cd4 clone 4sm95, by Thermo Fisher Scientific (1 mentions)
Biotinylated rabbit anti rat, by Dianova (1 mentions)
Envision system hrp labelled polymer anti rabbit, by Agilent Technologies (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Anti digoxigenin antibody, by Roche (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Asp175, by Cell Signaling Technology (1 mentions)
Power block, by BioGenex (1 mentions)
Aquatex, by Merck Group (1 mentions)
Avidin biotin blocking kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin ap, by Vector Laboratories (1 mentions)
Biotinylated secondary anti rabbit igg, by Vector Laboratories (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
Antifade reagent, by Cell Signaling Technology (1 mentions)
14 protocols using fast red
1
Immunohistochemical Analysis of Kidney
2
Quantifying Apoptosis by TUNEL Assay
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FACS data on apoptosis was further verified using the terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labelling (TUNEL) assay. The TUNEL technique was performed using the in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s instructions. Chamber slides were fixed with 4 % paraformaldehyde for 1 h, and 0.1 % Triton-100 in 0.1 % sodium citrate was added at 4 °C for 2 min. The slides were incubated with the TUNEL reaction mixture for 1 h at 37 °C. After washing, the slides were further incubated with alkaline phosphatase-conjugated anti-fluorescein antibody for 30 min at 37 °C. Slides were developed using Fast Red (DAKO, Carpenteria, CA, USA) and lightly counterstained with hematoxylin.TUNEL positive cells were observed under confocal microscopy. TUNEL-positive cells per field were counted in 5 random fields under 40× magnifications, and positive cell percentages were averaged.
3
Immunohistochemistry and in situ Hybridization Analysis of Brain Tissue
4
Immunohistochemical Analysis of Intestinal Tissue
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In situ immunohistochemical analysis of ileal and colonic paraffin sections was performed as previously described (Heimesaat et al., 2007 (link), 2010 (link); Alutis et al., 2015 (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), and B220 (eBioscience, 1:200) were used. For detection, the LSAB method was applied with FastRed (Dako) as chromogen. For each animal, the average number of positively stained cells within at least six high power fields (HPF, 400× magnification) was determined microscopically by a blinded investigator.
5
Immunohistochemical Analysis of TGF-β1 in IMCD Cells
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IMCD cells (5000 cells/well) were seeded on BD multichamber slides and underwent the gradual increase in osmolarity as described above. At 330, 600 and 900 mOsm, cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, cells were blocked using 1× Powerblock (Biogenex, San Ramon, CA, USA) for 10 min and incubated with rabbit polyclonal TGF-ß1 antibody (1:50 in PBS, Santa Cruz) overnight at 4C. The slide was carefully washed twice with PBS and incubated with secondary antibody (Rabbit Link, Biogenex) for 30 min, washed and developed with Fast Red (Dako). Nuclei were counterstained with hematoxylin solution, then slides were mounted using Aquatex (Merck) and analyzed under light microscope.
6
Immunohistochemical Detection of Rickettsia conorii
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The tissues were fixed with 10% neutral buffered formalin for two weeks and embedded in paraffin. The samples were sectioned at 5 μM thickness and stained with hematoxylin and eosin for histological analysis or processed for immunohistochemical (IHC) staining of rickettsial antigen [19 (link)].
The sections were incubated at 54°C overnight, deparaffinized and hydrated. After that, they were blocked with Avidin/Biotin Blocking Kit (Life Technologies, Frederick, MD) and treated with proteinase K (Dako, Carpinteria, CA), for antigen retrieval. The sections were incubated with polyclonal rabbit anti-R. conorii antibody (1:300 dilution, produced in-house) at room temperature for one hour, followed by biotinylated secondary anti-rabbit IgG (1:200 dilution, Vector Laboratories, Burlingame, CA), streptavidin-AP (1:200, Vector Laboratories, Burlingame, CA) for 30 minutes each and Fast Red (Dako, Carpinteria, CA) for 5 minutes. The sections were washed twice with Tris-buffered saline containing 0.05% Tween-20. Slides were counterstained with hematoxylin, dehydrated, mounted with Permount and examined with an Olympus BX51 microscope (Olympus Scientific, Waltham, MA).
The sections were incubated at 54°C overnight, deparaffinized and hydrated. After that, they were blocked with Avidin/Biotin Blocking Kit (Life Technologies, Frederick, MD) and treated with proteinase K (Dako, Carpinteria, CA), for antigen retrieval. The sections were incubated with polyclonal rabbit anti-R. conorii antibody (1:300 dilution, produced in-house) at room temperature for one hour, followed by biotinylated secondary anti-rabbit IgG (1:200 dilution, Vector Laboratories, Burlingame, CA), streptavidin-AP (1:200, Vector Laboratories, Burlingame, CA) for 30 minutes each and Fast Red (Dako, Carpinteria, CA) for 5 minutes. The sections were washed twice with Tris-buffered saline containing 0.05% Tween-20. Slides were counterstained with hematoxylin, dehydrated, mounted with Permount and examined with an Olympus BX51 microscope (Olympus Scientific, Waltham, MA).
7
Immunofluorescence and Immunohistochemistry of PLK4 in Keloids
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Cells were fixed and permeabilized with 0.1% Triton X‐100. The cells were blocked with 5% bovine serum albumin (BSA) and then immunostained with rabbit anti‐PLK4 antibody (1:200; Abcam), mouse anti‐Ki67 antibody (1:200; Abcam), or mouse anti‐γ‐tubulin antibody (1:200; Sigma‐Aldrich, USA), followed by incubation with a goat anti‐rabbit or goat anti‐mouse secondary antibody (1:400; Invitrogen), phalloidin (1:400; Abcam), and 4',6‐diamidino‐2‐phenylindole (1:800; Sigma‐Aldrich). Samples were mounted with an anti‐fade reagent (Cell Signaling Technology, USA). Cell area analysis was performed using ImageJ software. The relative cell areas were normalized to NFs. The immunofluorescence staining of KFs and NFs was conducted on at least three biological replicates.
Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were blocked with 5% BSA and incubated with a rabbit anti‐PLK4 antibody (1:200; Abcam). After sequential incubation with a biotinylated secondary antibody (Dako, Denmark) and an ABC‐alkaline phosphatase complex (Vector, USA), specific staining was revealed by using Fast Red (Dako). Immunohistochemistry staining of keloid and adjacent normal skin samples was conducted on at least three biological replicates.
Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were blocked with 5% BSA and incubated with a rabbit anti‐PLK4 antibody (1:200; Abcam). After sequential incubation with a biotinylated secondary antibody (Dako, Denmark) and an ABC‐alkaline phosphatase complex (Vector, USA), specific staining was revealed by using Fast Red (Dako). Immunohistochemistry staining of keloid and adjacent normal skin samples was conducted on at least three biological replicates.
8
Characterization of Adipose-Derived Stem Cells
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After 28 days of culture, Unpass-ASC were fixed with PFA 1% for 1 hour and washed twice with PBS. Immunohistochemical staining using antibodies against Collagen type I (MP Biomedicals, Illkirch-Graffenstaden, France) and fibronectin was performed as previously described8 (link). After incubation with a biotinylated secondary antibody and subsequently with an ABC-alkaline phosphatase complex, the specific staining was revealed by using Fast Red (All reagents from Dako, Baar, Switzerland). Matched IgG control antibodies were used as negative control.
For immunofluorescent staining, freshly harvested human fat tissue was frozen in liquid nitrogen, fixed in pure acetone for 10 minutes at −20 °C and then cryosectioned. Sections of 10 μm in thickness were stained with the following primary antibodies and dilutions: mouse anti-human CD49e (BD Biosciences, Basel, Switzerland) at 1:50; mouse anti-human; rabbit monoclonal anti-fibronectin (Abcam, Cambridge, UK) at 1:200; and rabbit polyclonal anti-laminin (Abcam) at 1:50. Fluorescent-conjugated secondary antibodies (Invitrogen, Basel, Switzerland) were used at 1:200. Images were acquired with a Nikon A1R confocal microscope.
For immunofluorescent staining, freshly harvested human fat tissue was frozen in liquid nitrogen, fixed in pure acetone for 10 minutes at −20 °C and then cryosectioned. Sections of 10 μm in thickness were stained with the following primary antibodies and dilutions: mouse anti-human CD49e (BD Biosciences, Basel, Switzerland) at 1:50; mouse anti-human; rabbit monoclonal anti-fibronectin (Abcam, Cambridge, UK) at 1:200; and rabbit polyclonal anti-laminin (Abcam) at 1:50. Fluorescent-conjugated secondary antibodies (Invitrogen, Basel, Switzerland) were used at 1:200. Images were acquired with a Nikon A1R confocal microscope.
9
Immunohistochemical Detection of CD74 and c-Met
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The detection of CD74 protein in formalin-fixed and paraffin-embedded tissue sections was performed employing the anti-CD74 antibodies sc-20062 and sc-6262 (both Santa Cruz) at a dilution of 1:1000 after a 20 min treatment in EDTA buffer (Retrieval solution 2; EDTA-buffer pH 8.8 at 98 °C for 20 min). Bound antibody was visualized using the alkaline phosphatase anti-alkaline phosphatase method and FastRed as chromogen (DAKO) or, after peroxidase blocking, DAB-chromogen. For c-MET detection, the prediluted anti-total c-Met (clone SP44) rabbit monoclonal antibody was obtained from Ventana (Ventana Medical System). Immunostaining was carried out according to the manufacturer´s protocol on the BenchMark Ultra platform from Ventana utilizing the ultraView detection kit.
10
Immunohistochemical Detection of Viral and Cytokine Distribution
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To detect viral distribution in tissues, IHC was performed on samples from rZJ1-IL2-, rZJ1-GFP, and mock-infected birds, as previously described [41 (link)]. Briefly, slides were deparaffinized, rehydrated, microwaved for 20 min at minimum power in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA) and blocked with an universal blocking reagent (Biogenex, San Ramon, CA). The primary antibody (polyclonal) was raised in rabbit against a synthetic peptide (TAYETADESETRRIC), which is highly conserved in NDV nucleoprotein, and used at 1:8000 dilution [42 (link)]. The detection system was an avidin–biotin–alkaline phosphatase system (Vector Laboratories, Burlingame, CA) coupled with a naphthol-based chromogen (Fast Red, Dako, Carpinteria, CA). Sections were counterstained lightly with hematoxylin and coverslipped with Permount for a permanent record. The scoring system for assessment of immunolabeling for NDV NP was previously published [41 (link)], and is reported in the caption of Table 2 . For IL-2, the same procedure was deployed; the chicken IL-2 antibody was a rabbit polyclonal (Kingfisher, Carlsbad, CA) used at 1:1000 dilution.
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