Indoleamine 2,3-dioxygenase expression was evaluated in the hippocampus and distal ileum. Briefly, paraffin sections were dewaxed, blocked with goat serum, and incubated with a rabbit anti-mouse indoleamine 2,3-dioxygenase antibody (1:40, ab106134, Abcam, Cambridge, UK) at 4°C overnight, and subsequently incubated with a goat anti-rabbit secondary antibody (1:1000, ab150077, Abcam, Cambridge, UK) for 30 min at room temperature. Sections were developed for imaging with 3,3'-diaminobenzidine and hematoxylin counterstain. Negative controls were incubated with PBS instead of antibodies. Indoleamine 2,3-dioxygenase-positive cells appeared as brown or yellow-brown particles. Ten fields of vision presenting dense positive particles were selected from the hippocampal CA1 region and distal ileum at low magnification and analyzed using Image-Pro Plus software (Nikon, Tokyo, Japan). Mean absorbance values were measured under high magnification (40X).
Ab106134
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab106134 is a laboratory equipment product manufactured by Abcam. It serves as a core function within the research and scientific community, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus software, by Nikon (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab203035, by Abcam (1 mentions)
Ab79056, by Abcam (1 mentions)
Ab 214214, by Abcam (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Ab5690, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Ab216485, by Abcam (1 mentions)
Rabbit anti c jun, by Cell Signaling Technology (1 mentions)
Rabbit anti p c jun, by Cell Signaling Technology (1 mentions)
Luminescent image analyzer lsa 4000, by GE Healthcare (1 mentions)
Ac026, by ABclonal (1 mentions)
3 3 diaminobenzidine (dab), by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab121013, by Abcam (1 mentions)
Enhanced chemiluminescence ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Biomiga (1 mentions)
6 protocols using ab106134
1
Indoleamine 2,3-dioxygenase Expression Quantification
2
Immunohistochemical Analysis of IDO and T-cell Markers
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Paraffin-embedded specimens were prepared using standard methods. Sections were stained with hematoxylin and eosin (H & E) and immunohistochemical staining. The primary Abs were anti-IDO1 (ab106134; Abcam, Cambridge, UK), anti-IDO2 (ab214214; Abcam), anti-CD3 (ab5690; Abcam), anti-CD4 (ab183685; Abcam), anti-CD8 (ab203035; Abcam), anti-IL-17 (ab79056; Abcam), and anti-Ki-67 (NB500-170; Novus Biologicals, Centennial, USA). Histofine Simple Stain MAX PO (Nichirei Biosciences, Tokyo, Japan) was used for mouse samples and Dako Envision System-Labeled Polymer HRP (Dako, Tokyo, Japan) for human samples as secondary antibodies.
3
Protein Extraction and Western Blot Analysis
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Cells and tissues were lysed with RIPA buffer, protein were extracted and quantitated, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a PVDF membrane. The protein containing membrane was washed, incubated with the indicated primary antibodies, washed, and further incubated with secondary antibodies. After washing with PBS, the proteins on the membrane was then detected by electrochemiluminescence (ECL) in a Luminescent Image Analyzer LSA 4000 (GE, Fairfield, CO, USA). The antibodies used in this study included rabbit anti-β-Actin (AC026, Abclonal, USA; 1:50000), rabbit anti-c-Jun (#9165S, Cell Signaling Technology, USA, 1:500), rabbit anti-p-c-Jun (#9261S, Cell Signaling Technology, 1:500), rabbit anti-α7nAChR (#ab216485, Abcam, Cambridge, MA, USA; 1:500) and rabbit anti-IDO1 (ab106134, Abcam; 1:500) antibodies.
4
Immunohistochemical Analysis of IDO1 Expression
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Immunohistochemistry was used to analyze IDO1 expression in the tumor tissue. Paraffin sections were kept for 30 minutes in an oven at 60°C temperature and then submitted to the process of deparaffinization and rehydration by successive baths in xylol and alcohol. Antigenic recovery was performed using steam-heated EDTA buffer, followed by peroxidase blockage using a 3% hydrogen peroxide solution. To increase nonspecific binding blockade, samples were subjected to a final block consisting of 6% rehydrated milk (Nestlé Brasil LTDA, Sao Paulo, Brazil) and 0.5% bovine serum albumin for 15 minutes. After removal of the excess blockage volume, sections were incubated with anti-IDO1 antibody (ab106134, Abcam, Cambridge, MA, USA) diluted 1:10 in the previous blocking solution and kept overnight in a humid chamber in the refrigerator at 4°C. To complete the sandwich, sections were incubated with EnVision Flex HRP reagents (K0690; Dako Co, Denmark) following manufacturer recommendations. DAB chromogen substrate was used to complete the reaction (K346811; Dako Co, Denmark). As a final step, sections were stained with Harris’ hematoxylin.
5
Immunohistochemical Analysis of IDO1 Expression
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Immunohistochemistry (IHC) assays were performed in lung tissue samples isolated from patients and mice. Tumor tissues were fixed with formalin, embedded in paraffin, and cut into 3–5 μm slides. The slides were then deparaffinized with xylene and graded alcohol, and underwent a heat-induced epitope retrieval step in citrate buffer solution. The slides were blocked with 5% BSA, incubated with an anti-IDO1 antibody (ab106134, Abcam, USA; 1:50), and then incubated with an HRP-labeled goat anti-mouse IgG antibody. Immunoreactions were evaluated with the 3,3′-diaminobenzidine (from Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and hematoxylin. The percentage of staining-positive cells (RP) was evaluated, the staining intensity (SI) was determined, and the immunoreactivity score (IRS) was calculated as IRS (0–12) = RP (0–4) × SI (0–3).
6
Western Blot Analysis of TPH2 and IDO1
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The expression of the TPH2 and IDO1 proteins in the hippocampus was detected by Western blot. The hippocampal tissues were used to prepare the total proteins with RIPA Lysis buffer (Biomiga, Santiago, CA, USA). The 10% SDS-PAGE gels were selected according to relative molecular weight of TPH2 (56 kDa) and IDO1 (45 kDa), and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The 5% nonfat milk was used to block the membranes, and the primary antibodies were anti-TPH2 antibody (ab121013, goat polyclonal to TPH2, diluted 1:500; Abcam, San Francisco, CA, USA), anti-indoleamine 2,3-dioxygenase antibody (ab106134, rabbit polyclonal to indoleamine 2,3-dioxygenase, diluted 1:50; Abcam) and β-actin monoclonal antibody (MA1-140, mouse monoclonal to β-actin, diluted 1:3,000; Thermo Fisher Scientific). The enhanced chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific) was used to develop the membranes for 1 minute, and then the Tanon-5200 system (Tanon, Shanghai, China) was used for exposure. The optical density of protein bands was read by Tanon Gis software (Tanon).
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