Formalin-fixed paraffin-embedded samples were sectioned at 4 μm. After antigen retrieval by autoclaving in tris-ethylenediaminetetraacetic acid based buffer (S2375; Dako, Carpinteria, CA, USA), sections were incubated with 3% hydrogen peroxide and protein-free blocking solution (X0909; Dako). Sections were stained with rabbit anti-human CD117 (1:500, A4502; Dako), rabbit anti-human CD3 (1:150, ab16669; Abcam, Cambridge, UK) or mouse anti-human Forkhead box P3 (FOXP3) (1:500, ab20034; Abcam), sequentially. Sections were then incubated with EnVision+ Dual Link System-horseradish peroxidase (HRP) (K4063; Dako), EnVision rabbit-HRP (K4003; Dako) or EnVision mouse-HRP (K4001; Dako) and developed with 3,3´-Diaminobenzidine substrate using the chromogen system (K3468; Dako). Finally, specimens were counterstained with Mayer’s hematoxylin. The peak mast cells, T cells and Tregs counts were obtained by counting at least 3 representative non-overlapping HPFs using a microscope (Olympus BX51; Tokyo, Japan) at 400× magnification in a blinded manner. Samples were evaluated by JC and TO in a blind manner. In the event of discordance in histological evaluation, the specimen underwent further review until a consensus was reached on the assessment.
S2375
Manufactured by Agilent Technologies
Sourced in United States
The S2375 is a high-performance microwave generator from Agilent Technologies. It is designed to provide stable and precise microwave signals for a variety of laboratory and research applications.
Automatically generated - may contain errors
Lab products found in correlation
A4502, by Agilent Technologies (2 mentions)
X0909, by Agilent Technologies (2 mentions)
Ab16669, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
K3468, by Agilent Technologies (1 mentions)
Envision dual link system horseradish peroxidase hrp, by Agilent Technologies (1 mentions)
Clone ae1 ae3, by Agilent Technologies (1 mentions)
Ncl l pgr 312, by Leica (1 mentions)
Ncl l er 6f11, by Leica (1 mentions)
Biotinylated anti igg secondary antibodies, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Sr p37, by Panasonic (1 mentions)
Ab109493, by Abcam (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
4 protocols using s2375
1
Immunohistochemical Profiling of Immune Cells
2
Immunohistochemical Analysis of Tumor Samples
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Tumor and metastatic samples were fixed in 4% paraformaldehyde and embedded in paraffin. Antigen retrieval was performed using pH9 antigen retrieval buffer (DAKO S2375) at 95 °C for 20 min or citrate buffer pH6 at 95 °C for 20 min for CC3. Antibodies against ER (NCL-L-ER-6F11, Novocastra), PR (NCL-L-PGR-312, Novocastra), HER2 (SP3, Spring Bioscience), or pan-cytokeratin (DAKO, Clone AE1/AE3) were used at 4 °C overnight, followed by biotinylated anti-IgG secondary antibodies (Vector Labs). The signal was detected by incubation with ABC Elite (Vector Labs) for 30 min and 3,3′-diaminobenzidine (Dako) for 5 min at room temperature.
3
Quantifying Tumor-Infiltrating Lymphocytes and FOXP3
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The paraffin sections were rehydrated and immersed in 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Epitope retrieval was performed by heating the sections at 95 °C in Target Retrieval Solution (pH 9, S2375; Dako) for 30 min in an electric pressure cooker (SR-P37; Panasonic, Tokyo, Japan). All incubations after the blocking step were performed at room temperature. The wash buffer was 0.05 M Tris-buffered saline with 0.05% Tween 20 (pH 7.6). Following blocking with 5% normal goat serum (Abcam) in PBS for 10 min, the sections were incubated with anti-FOXP3 antibodies (236 A/E7; Abcam) for 1 h.
Histopathologic assessment of the percentage of TILs and FOXP3 was performed on one representative immunohistochemical section of a tumor using methods recommended by the International TILs Working Group 201418 (link). % TILs was defined as the percentage of lymphocyte area per tumor area in vivo samples. Areas of in situ carcinomas, normal lobules, necrosis, hyalinization, and crush artifacts were not included. Histopathologic evaluation of TILs was performed by two breast pathologists who scored each case independently in a blind manner. The mean values of both observers were obtained as the final scores for each case.
Histopathologic assessment of the percentage of TILs and FOXP3 was performed on one representative immunohistochemical section of a tumor using methods recommended by the International TILs Working Group 201418 (link). % TILs was defined as the percentage of lymphocyte area per tumor area in vivo samples. Areas of in situ carcinomas, normal lobules, necrosis, hyalinization, and crush artifacts were not included. Histopathologic evaluation of TILs was performed by two breast pathologists who scored each case independently in a blind manner. The mean values of both observers were obtained as the final scores for each case.
4
Immunohistochemical and Immunofluorescent Staining of Mast Cells and IgG4 in Esophageal Biopsies
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Paraffin-embedded, 4 µm-thick sections of biopsy specimens were deparaffinized as mentioned above. After antigen retrieval by autoclaving in tris-EDTA-based buffer (S2375; Dako, Carpinteria, CA, USA), non-specific binding was blocked with 3% hydrogen peroxide and protein blocking solution (X0909; Dako) for immunohistochemical staining. Mast cells were stained with rabbit anti-human CD117, c-kit (1:500; A4502, Dako) for 30 min at room temperature. Specimens were then sequentially incubated with secondary EnVision Dual Link System-HRP® (K4063; Dako) for 30 min at room temperature and the chromogen diaminobenzidine, then counterstained with Mayer’s hematoxylin. Mast cells were counted in at least three representative non-overlapping HPFs at 400× magnification in a blinded manner.
Non-specific binding was blocked with 5% bovine serum albumin for immunofluorescent staining. IgG4 was stained with rabbit anti-human IgG4 antibody (1:1000; AB109493, Abcam, Cambridge, UK) for 60 min at room temperature. Specimens were then incubated with Cy3-conjugated goat anti-rabbit IgG (A120-201C3; Bethyl Laboratories, Montgomery, TX, USA) and DAPI for 30 min at room temperature. Slides were examined under a confocal laser-scanning microscope (LSM780). IgG4 deposits were defined as the existence of granular intercellular staining in the esophageal epithelia [17 (link)].
Non-specific binding was blocked with 5% bovine serum albumin for immunofluorescent staining. IgG4 was stained with rabbit anti-human IgG4 antibody (1:1000; AB109493, Abcam, Cambridge, UK) for 60 min at room temperature. Specimens were then incubated with Cy3-conjugated goat anti-rabbit IgG (A120-201C3; Bethyl Laboratories, Montgomery, TX, USA) and DAPI for 30 min at room temperature. Slides were examined under a confocal laser-scanning microscope (LSM780). IgG4 deposits were defined as the existence of granular intercellular staining in the esophageal epithelia [17 (link)].
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