Multiplex staining was performed following the Opal Tyramide amplification signal protocol for the markers D2–40 (D2/40; Abcam, Cambridge, MA, USA) labeled with fluorophore 520 (Perkin Elmer, Akron, OH, USA); CD68 (KP1; Agilent, Santa Clara, CA, USA) labeled with fluorophore 520; and CD206 (5C11; LSBio, Seattle, WA, USA) labeled with fluorophore 570 (Perkin Elmer). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Perkin Elmer) and cover slips were applied to the slides with ProLong Gold (Life Technologies, Grand Island, NY, USA). The markers were distributed between two different panels of three colors each. The first panel included D2–40 and CD206; the second, CD68 and CD206. Slides containing prostate sections were deparaffinized in xylene and rehydrated as described above. For antigen retrieval, slides were microwaved with citrate H25 pH 6.0 buffer at 100 W for 1 min and 20 W for 15 min. After cooling for 15 min, they were treated with dual horseradish peroxidase block for 10 min, and the primary and secondary antibodies and fluorophores were applied according to their respective dilutions and incubation periods in a humid chamber. For the second round of primary antibody application, slides were first subjected to the same microwave antigen retrieval steps described above.
D2 40
Manufactured by Abcam
Sourced in United Kingdom, United States
D2-40 is a monoclonal antibody that specifically binds to the podoplanin protein, which is a marker for lymphatic endothelial cells. This antibody can be used to detect and identify lymphatic vessels in various tissue samples.
Automatically generated - may contain errors
3 protocols using d2 40
1
Multiplex Immunofluorescence Staining for Cell Markers
2
Podoplanin Expression and Lymphatic Vessel Scoring
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An antibody detecting podoplanin (D2‐40; Abcam, Cambridge, UK) was diluted 1:25. Slides were pretreated in TRIS‐EDTA pH 8.0, and staining was performed in a Ventana staining machine according to the supplier's recommendations. The percentage of tumour cells expressing podoplanin as well as the intensity of staining was assessed. The percentage of tumour cells expressing podoplanin was divided into six groups, where 0–4% = 1, 5–19% = 2, 20–39% = 3, 40–59% = 4, 60–79% = 5 and 80–100% = 6. Staining intensity in turn was described as negative = 0, weak = 1, intermediate = 2 or strong = 3. By multiplying the score for percentage of podoplanin‐expressing tumour cells with the score for staining intensity, a quick score (QS) was calculated for each slide 9 . Scoring of slides was performed independently by three of the authors (NS, ELJ and KN) and cases of disagreement were reevaluated and discussed to provide a consensus score.
The number of lymph vessels in each sample was scored between 0 and 3, with 0 being no lymph vessels detectable, 1 = few, 2 = moderate numbers and 3 = many lymph vessels detectable, respectively.
The number of lymph vessels in each sample was scored between 0 and 3, with 0 being no lymph vessels detectable, 1 = few, 2 = moderate numbers and 3 = many lymph vessels detectable, respectively.
3
Podoplanin Immunostaining for Lymphatic Mapping
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A 200 section series was cut on a paraffin microtome at a thickness of 5 µm for both lung samples. Every 10th section was immunostained for lymphatic endothelial cells. This was achieved by using D240 (Abcam, Cambridge UK: ab77854) mouse monoclonal anti-human podoplanin primary antibody following a standard immunohistochemistry protocol. To confirm specific staining of the D240 antibody, anti-CD31 (Abcam: ab28364) and anti-pan-keratin (Sigma-Aldrich Company Ltd., Dorset, England: C-2562) were used to stain blood vessels and airways respectively on serial sections (see supplementary material page 1). Negative controls (omitting primary antibody) were included in each staining run.
An Olympus dotslide (Olympus, Southend-on-Sea, UK) system automatically obtained multiple images of the D240 stained slides at 20X magnification and digitised the resulting image as a VSI file. OlyVia software (Olympus) was used to view the slide images.
An Olympus dotslide (Olympus, Southend-on-Sea, UK) system automatically obtained multiple images of the D240 stained slides at 20X magnification and digitised the resulting image as a VSI file. OlyVia software (Olympus) was used to view the slide images.
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