Primary cells were grown on 12-mm glass coverslips in 24-well plates. Cells were fixed for 10 min with 4% paraformaldehyde and permeabilized with 0.05% saponin for 5 min. The cells were then incubated over night at 4°C with Calretinin (N-term) (Sigma HPA007306), Podoplanin (D2-40) (Dako M3619), YAP1 (Cell Signaling 4912), Ki67 (Abcam ab8191), Phalloidin 488 (molecular probes A12379), Thy1 (H-110) (Santa Cruz sc-9163), and N-Cadherin from Dako (M3613) diluted in PBS containing 1% bovine serum albumin. Secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies A11034) and Alexa Fluor 555-conjugated goat anti-mouse IgG (Life Technologies A21424) antibody was added for 1 h at RT. Nuclear DNA was stained using DAPI. Coverslips were mounted using Prolong Gold antifade reagent (Life Technologies). Images were acquired using an Olympus BX61 microscope (Schwerzenbach, Switzerland) equipped with an F-view camera for conventional fluorescence imaging. The image capture was controlled with the AnalySISPro software (Soft Imaging System, Münster, Germany).
Podoplanin d2 40
Manufactured by Agilent Technologies
Sourced in Denmark
Podoplanin (D2-40) is a laboratory reagent used for the identification and differentiation of lymphatic endothelial cells. It is a transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells, as well as in certain other cell types. This product can be used in immunohistochemical and immunocytochemical applications to detect the presence and distribution of podoplanin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Agilent Technologies (1 mentions)
Thy1 h 110, by Santa Cruz Biotechnology (1 mentions)
Analysis pro software, by Olympus (1 mentions)
Phalloidin 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Novared, by Vector Laboratories (1 mentions)
Deep space black, by Biocare Medical (1 mentions)
Glycerol gelatin aqueous slide mounting medium, by Merck Group (1 mentions)
Von willebrand factor, by Agilent Technologies (1 mentions)
Permared ap, by Diagnostic BioSystems (1 mentions)
Roti histokitt, by Carl Roth (1 mentions)
Ae1 3, by Agilent Technologies (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
3 protocols using podoplanin d2 40
1
Immunofluorescence Staining of Primary Cells
2
Immunohistochemical Staining of Mouse Proteins
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The following primary mouse anti-human protein antibodies (clone; manufacturer)
were used in this study: RORγt (6F3.1; Merck Millipore, Darmstadt, Germany), CD3
(A-1; Santa Cruz Biotechnology, CA), CD20 (L26; Immunologic, Duiven,
Netherlands), CD79α (JCB117; Dako, Glostrup, Denmark), and Podoplanin (D2-40;
Dako). The following rabbit anti-human protein antibodies were used: T-bet/Tbx21
(EPR9302; Abcam, Cambridge, UK), GATA3 (EPR16651; Abcam), CD3 (SP7;
Immunologic), CD56 (MRQ-42; Cell Marque, Rocklin, CA), and von Willebrand Factor
(polyclonal; Dako).
As secondary reagents we used alkaline phosphatase (AP)- or horseradish
peroxidase (HRP)-conjugated anti-mouse and anti-rabbit BrightVision detection
kits (Immunologic). Chromogens PermaBlue/AP, PermaRed/AP, and PermaGreen/HRP
were from Diagnostic Biosystems (Sanbio, Netherlands), Deep Space Black was
purchased from Biocare Medical (Concord, CA) and Nova-RED was from Vector
Laboratories (Burlingame, CA). Antibodies and polymers were always diluted in
Normal Antibody Diluent from Scytek Laboratories (Logan, UT). We used
Tris-buffered saline solution with 0.05% Tween (TBST) as washing buffer between
incubation steps. The stained sections were mounted in Glycerol Gelatin aqueous
slide mounting medium (Sigma-Aldrich, Zwijndrecht, Netherlands) or in
PertexTM (VWR international, Amsterdam, Netherlands).
were used in this study: RORγt (6F3.1; Merck Millipore, Darmstadt, Germany), CD3
(A-1; Santa Cruz Biotechnology, CA), CD20 (L26; Immunologic, Duiven,
Netherlands), CD79α (JCB117; Dako, Glostrup, Denmark), and Podoplanin (D2-40;
Dako). The following rabbit anti-human protein antibodies were used: T-bet/Tbx21
(EPR9302; Abcam, Cambridge, UK), GATA3 (EPR16651; Abcam), CD3 (SP7;
Immunologic), CD56 (MRQ-42; Cell Marque, Rocklin, CA), and von Willebrand Factor
(polyclonal; Dako).
As secondary reagents we used alkaline phosphatase (AP)- or horseradish
peroxidase (HRP)-conjugated anti-mouse and anti-rabbit BrightVision detection
kits (Immunologic). Chromogens PermaBlue/AP, PermaRed/AP, and PermaGreen/HRP
were from Diagnostic Biosystems (Sanbio, Netherlands), Deep Space Black was
purchased from Biocare Medical (Concord, CA) and Nova-RED was from Vector
Laboratories (Burlingame, CA). Antibodies and polymers were always diluted in
Normal Antibody Diluent from Scytek Laboratories (Logan, UT). We used
Tris-buffered saline solution with 0.05% Tween (TBST) as washing buffer between
incubation steps. The stained sections were mounted in Glycerol Gelatin aqueous
slide mounting medium (Sigma-Aldrich, Zwijndrecht, Netherlands) or in
PertexTM (VWR international, Amsterdam, Netherlands).
3
Histological Assessment of Lymphatic Vessels
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In order to assess the histology of the lymph vessel biopsies, sections flanking the tissue for RNA extraction were stained for the epithelial cells (AE1/3) and lymphatic cells (D2‐40). Briefly, cryosections of 7 μm thickness were fixed on Superfrost Plus slides with 4% paraformaldehyde for 10 minutes. Slides were washed with tris‐buffered saline (50 mM Tris, 150 mM NaCl) and 0.1% Tween‐20 (TBST) and incubated in 0.6% H2O2 for 7 minutes. Staining was performed for 1 hour at room temperature with AE1/3 (Dako) diluted to a final concentration of 1.7 μg/mL and podoplanin D2‐40 (Dako) diluted to a final concentration of 0.5 μg/mL. For detection, the DAKO EnVision System was used according to the manufacturer's protocol. Finally, slides were counterstained with hematoxylin, dehydrated in alcohol, equilibrated in xylol, and embedded using RotiHistokitt (Roth).
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