IHC staining was used to evaluate the intratumoral and peritumoral presence of TAMs (CD68-positive) and M2-like macrophages (CD163-positive), a subtype of TAM, in primary and metastasis site tissues. Five micrometer-thick sequential tissue sections were obtained from paraffin-embedded tissue samples and used for the IHC analysis as described in previous studies[15 (link),16 (link)]. The primary antibodies used in IHC staining were antibodies against CD68 (rabbit anti-human, D4B9C, dilution 1:400; Cell Signaling Technology, Danvers, MA, United States) and CD163 (rabbit anti-human, D6U1J, dilution 1:500; Cell Signaling Technology). Anti-rabbit IgG, horseradish peroxidase-linked antibody was used as the secondary antibody. For the assessment of expression of CD68- and CD163-positive cells, the tissue sections were screened using each immunohistochemistry slide at the low power fields (× 4), and the hot spots were selected. Immune cell staining was scored by counting the number of stained immune cells in three high power fields (× 400) in the hot spots. IHC sections were independently assessed by three authors (Yang J, Chen YH, and Zhou L).
D6u1j
Manufactured by Cell Signaling Technology
Sourced in United States
D6U1J is a lab equipment product from Cell Signaling Technology. It is a microplate reader designed for quantitative analysis of various biochemical and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
D4b9c, by Cell Signaling Technology (1 mentions)
Anti cd68, by Thermo Fisher Scientific (1 mentions)
Anti cd163 antibody, by Cell Signaling Technology (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
Anti cd86, by Cell Signaling Technology (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
2 protocols using d6u1j
1
Quantifying Tumor-Associated Macrophages in Tissues
2
Immunohistochemical Analysis of Tumor Infiltrates
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Tumor biopsies were fixed in 3.7% neutral-buffered formaldehyde and embedded in paraffin according to standard protocols. Two-micrometer sections were prepared, and morphology was visualized by standard H&E staining. For immunohistochemistry, the activity of the endogenous peroxidase was blocked with 1% hydrogen peroxide, and after antigen retrieval (citric acid buffer, pH 6) at 100°C, sections were incubated with anti-CD68 (dilution 1:250, clone KP1, ThermoFisher Scientific, Waltham, MA, USA), anti-CD86 (dilution 1:75, clone E2G8P, Cell Signaling Technology, Beverly, CA, USA), and anti-CD163 antibody (dilution 1:250, clone D6U1J, Cell Signaling Technology, Beverly, CA, USA), respectively. This was followed by incubation with EnVision™+Dual Link System-HRP (Dako, Carpinteria, CA, USA). Diaminobenzidine (Cell Signaling Technology, Beverly, CA, USA) was used as a chromogen. Sections were counterstained with 1% Mayer’s hematoxylin. Slides were analyzed using a Leica dm2500 microscope equipped with LAS version 4 software (Leica, Germany).
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