Splenocytes were collected from naïve C57Bl/6N wild-type mice after lysis of red blood cells with PharmLyse buffer (BD Bioscience) and were labeled with 5 mM of CFSE (Sigma-Aldrich) in PBS for 15 min at 37°C. Then, 2 × 106 labeled splenocytes were infused into the tail vein of sex-matched recipient mice. Inguinal LNs were collected 30 min later, fixed with 10% paraformaldehyde overnight at room temperature, and embedded in paraffin. Seven-μm and 40-μm sections were deparaffinized, followed by antigen retrieval (10 mM citrate buffer [pH 6.0]) and immunofluorescence staining using goat anti-LYVE1 (R&D) and rabbit anti-ANXA2 (Abcam) antibodies and donkey anti-goat and anti-rabbit Alexa594- and Alexa647-conjugated secondary antibodies. Images covering all cortical and medullary regions were captured with an LSM 780 upright confocal microscope and analyzed with Fiji. The number of CFSE-labeled cells in ANXA2+ sinuses and in ANXA2- sinuses were counted manually using 7-μm sections. Maximum intensity projections of confocal z-stack images were prepared using 40-μm sections of the same samples.
Goat anti lyve 1
Manufactured by R&D Systems
Sourced in United States
Goat anti-LYVE-1 is a primary antibody that recognizes the LYVE-1 protein. LYVE-1 is a lymphatic endothelial hyaluronan receptor.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti cd31, by BD (3 mentions)
Goat anti prox1, by R&D Systems (3 mentions)
Lsm 710 fcs confocal microscope, by Zeiss (3 mentions)
Hamster anti podoplanin, by Abcam (3 mentions)
Rabbit anti rfp, by Rockland Immunochemicals (2 mentions)
Rat anti cd68, by Abcam (2 mentions)
Rabbit anti prox1, by Abcam (2 mentions)
Zen software, by Zeiss (2 mentions)
Rat anti endomucin, by Santa Cruz Biotechnology (2 mentions)
Chicken anti gfp, by Abcam (2 mentions)
Pharm lyse buffer, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Alexafluor 488 and 647 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Lavision ultramicroscope, by Miltenyi Biotec (1 mentions)
Rat anti f4 80, by Abcam (1 mentions)
Goat anti cd206, by R&D Systems (1 mentions)
Mouse anti smooth muscle actin, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Mouse anti cd163, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd68, by Thermo Fisher Scientific (1 mentions)
12 protocols using goat anti lyve 1
1
Quantifying Lymph Node Homing of Labeled Splenocytes
2
Lymph Node Clearing and Imaging
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Mice were sacrificed and lymph nodes were dissected and fixed in 4% PFA. After a wash in PBS, samples were permeabilized in 0.5% Triton-X 100 in PBS. After a wash in PBS, samples were blocked in blocking solution (1% BSA, 0.1% Tween-20 in PBS) and subsequently incubated with primary antibodies diluted in blocking solution. The antibodies used were rabbit anti-RFP (1:100, Rockland) and goat anti-LYVE-1 (1:100, R&D Systems). Samples were washed in PBS 0.1% Tween-20 (PBS-T) and incubated with AlexaFluor 488 and 647-conjugated secondary antibodies (1:200, Invitrogen) in blocking solution. After a wash in PBS-T, samples were embedded in 1% ultrapure low melting point agarose, dehydrated with methanol and cleared with BABB (benzyl alcohol/ benzyl benzoate 1:2) [20 (link)]. Briefly, samples were dehydrated in a series of 50%, 70%, 95% (at least 1 hour each) and 100% methanol (overnight) and cleared for 1 hour in 50% BABB in methanol and finally in BABB for at least 8 hours. Samples were stored in BABB at 4°C in the dark until image acquisition. Images were acquired using a LaVision Ultramicroscope (LaVision BioTec, Bielefeld). Stacks were captured using 2 μm step size and 2.5x magnification. Maximum projection of a 260 μm z-stack was obtained using the ImageJ software.
3
Immunofluorescence Staining of Lymphatic and Vascular Markers
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Tail skin and diaphragms were fixed in fresh 4 % paraformaldehyde (PFA) for 2 h and blocked at room temperature for 3 h in phosphate-buffered saline (PBS) containing 5 % donkey serum, 1 % bovine serum albumin (BSA) and 0.1 % Triton-X. The following antibodies were incubated overnight: Rabbit anti-LYVE-1 (AngioBio), goat anti-LYVE-1 (R&D Systems), rat anti-CD31 (BD Biosciences Pharmingen), goat anti-CD206 (R&D Systems), mouse anti-smooth muscle actin (Sigma-Aldrich), rat anti-CD68 (Abcam), goat anti-Prox-1 (R&D Systems), rabbit anti-Prox-1 (kind gift of Dr. Kari Alitalo), rat anti-F4/80 (Abcam) and rat anti-CSF-1R (AFS98 hybridoma antibody). After an intense washing step, tissues were incubated for 2 h at room temperature with Alexa Fluor 488, 594 or 647 nm-conjugated secondary antibodies (Invitrogen) and mounted in Vectashield (Vector) for confocal imaging. Whole-mount z-stack images were acquired with a LSM 710 FCS confocal microscope and ZEN software (Zeiss) and processed with Photoshop CS5 (Adobe) and ImageJ software (NIH).
4
Comprehensive Immunohistochemical Analysis of Lymph Nodes
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Immunohistochemistry was performed as previously indicated [73 (link), 82 (link)] on formalin-fixed paraffin-embedded (FFPE) LNs obtained at necropsy. Briefly, sections were deparaffinized and antigen retrieval was performed using a Retriever (Electron Microscopy Services, Hatfield, PA) in Tris-EDTA-Tween-80 buffer 73. Sections were stained for Tcells/B cells/dendritic cells (polyclonal rabbit anti-CD3, Dako, Santa Clara, CA; polyclonal rabbit anti-CD20, Thermo Fisher Scientific, Pittsburgh, PA; mouse-anti-CD11c, Leica Microsystems, Buffalo Grove, IL), macrophage subsets (mouse anti-CD68, Thermo Fisher; rabbit anti-DC-SIGN, ProSci Inc, Poway, CA; mouse anti-CD163, Thermo Fisher), LN vascular and structural aspects (Goat anti-LYVE-1, R&D Systems, Minneapolis, MN; rat-anti PNAd, BioLegend, San Diego, CA), and LN conduit systems (visualized by staining for rabbit anti-collagen 1 [Abcam, Cambridge, MA]). Primary antibodies were visualized with species- and isotype-specific secondary antibodies purchased from Jackson ImmunoResearch (West Grove, PA). Auramine rhodamine was performed as previously indicated [73 (link)] using reagents from BD Biosciences (San Jose, CA). Images were acquired at 20x magnification with a Nikon e1000 widefield microscope (Nikon, Melville, NY) with Nikon Elements.
5
Immunofluorescent Analysis of Meningeal Vasculature
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Meninges were fixed overnight in 4% paraformaldehyde while still attached to the
skullcap. Skullcaps were then incubated in ethanol/acetone for 20 min at −20℃ and then the
meninges were carefully dissected away from the skullcap. Meninges were washed with PBS
and then blocked overnight with PBS + 0.3% TX-100 + 20% Aquablock (East Coast Bio, North
Berwick, ME, USA). Primary antibody was then added and the meninges were incubated
overnight at 4℃. The following primary antibodies were used: rat anti-CD31 (1:1000;
eBiosciences, San Diego, CA, USA), goat anti-Lyve-1 (1:1000; R&D Systems, Minneapolis,
MN, USA), hamster anti-podoplanin (1:1000; Abcam, San Francisco, CA, USA), and rabbit
anti-Prox1 (1:500; AngioBioCo, Del Mar, CA, USA). Tissues were washed with PBS + 0.3%
TX-100 and then incubated overnight at 4℃ with the appropriate secondary antibodies.
Tissues were washed again with PBS + 0.3% TX-100, placed on slides, and coverslips were
mounted with ProLong Gold (Thermo Fisher Scientific, Waltham, MA, USA) anti-fade
reagent.
skullcap. Skullcaps were then incubated in ethanol/acetone for 20 min at −20℃ and then the
meninges were carefully dissected away from the skullcap. Meninges were washed with PBS
and then blocked overnight with PBS + 0.3% TX-100 + 20% Aquablock (East Coast Bio, North
Berwick, ME, USA). Primary antibody was then added and the meninges were incubated
overnight at 4℃. The following primary antibodies were used: rat anti-CD31 (1:1000;
eBiosciences, San Diego, CA, USA), goat anti-Lyve-1 (1:1000; R&D Systems, Minneapolis,
MN, USA), hamster anti-podoplanin (1:1000; Abcam, San Francisco, CA, USA), and rabbit
anti-Prox1 (1:500; AngioBioCo, Del Mar, CA, USA). Tissues were washed with PBS + 0.3%
TX-100 and then incubated overnight at 4℃ with the appropriate secondary antibodies.
Tissues were washed again with PBS + 0.3% TX-100, placed on slides, and coverslips were
mounted with ProLong Gold (Thermo Fisher Scientific, Waltham, MA, USA) anti-fade
reagent.
6
Immunofluorescence Staining of Lymphatic Markers
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The following antibodies were used: goat anti-LYVE-1 (polyclonal, R&D, AF2089), rat anti-PECAM-1 (clone 5D2.6 and IG5.1, provided by D. Vestweber, Max Planck Institute for Molecular Biomedicine Munster), rabbit anti-LYVE-1 (polyclonal, Reliatech), goat anti-LDLR (polyclonal, R&D, AF2255), rabbit anti-GS (polyclonal, Sigma-Aldrich, G2781), rabbit anti-CK19 (polyclonal, proteintech, 14965-1-AP), mouse anti-smooth muscle actin (SMA)-Cy3 (clone 1A4, Sigma), rat anti-Lyve-AlexaFluor594 (clone 223322, R&D), secondary antibodies coupled to AlexaFluor488, 568 and 647 (Invitrogen).
7
Lymphatic Vessel Visualization
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The paraffin sections were deparaffinized, rehydrated, and stained with the lymphatic marker goat anti-LYVE1 (R&D Systems, AF2125, Minneapolis, MN, USA) in a dilution of 1:100 and anti-goat secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A11055, Waltham, MA, USA) in a dilution of 1:250. Nuclear staining with 4′,6-Diamidino-2-phenylindole (DAPI) helped to visualize the gross morphology of the section. Images were acquired with a Nikon upright microscope connected to a camera.
8
Tumor Immunohistochemistry and Immunofluorescence
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The following primary antibodies were used for immunohistochemistry or immunofluorescence staining of tumors: goat anti-LYVE-1 (R&D Systems, cat no. AF2125), rat anti-endomucin (Santa Cruz, cat no. sc-65495), Cy3-conjugated mouse anti-smooth muscle actin (Sigma, cat no. C6198), and hamster anti-podoplanin (abcam, cat no. ab11936). All secondary antibodies were purchased from Jackson ImmunoResearch.
9
Cryosectioning and Immunostaining of Mouse Back Skin
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Back skin samples were embedded in OCT (Leica Biosystems, Newcastle, UK) and frozen in liquid nitrogen. 10-μm frozen sections were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT), washed in PBS and incubated with blocking solution (5% donkey serum, 0.2% BSA and 0.3% Triton X-100 in PBS) for 1 h at RT. Next, the sections were stained with primary antibodies overnight at 4°C and, after several washes, incubated with secondary antibodies for 30 min at RT. Primary antibodies were as follows: rat anti-CD31 (BD Biosciences, San Jose, CA, USA), rabbit anti-LYVE-1 (Angiobio, Del Mar, CA, USA), goat anti-LYVE-1 (R&D Systems), rat anti-CD68 (Abcam, Cambridge, MA, USA), goat anti-podoplanin (R&D Systems), rabbit anti-cytokeratin 15 (Abcam), rat anti-Foxp3 (eBioscience, San Diego, CA, USA), and goat anti-Prox1 (R&D systems). Secondary antibodies (all from Thermo Fisher, San Jose, CA, USA) were as follows: donkey anti-rabbit Alexa Fluor 488, 594 or 647, donkey anti-rat Alexa Fluor 488 or 594, donkey anti-goat Alexa Fluor 488 or 594, and chicken anti-goat Alexa Fluor 647. Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) was used for nuclear staining. Immunofluorescence images were acquired by an Axioskop 2 mot plus microscope (Carl Zeiss, Jena, Germany) and Z stacks of images were obtained using a Zeiss LSM 710 FCS confocal microscope.
10
Immunohistochemical Staining for Lymphatic Markers
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The following primary antibodies were used for immunohistochemistry or immunofluorescence staining: goat anti-Lyve1 (R&D Systems, AF2125; dilution 1:250 and 1:1000), chicken anti-GFP (Abcam, ab13970; dilution 1:1000), hamster anti-podoplanin (Abcam, ab11936; dilution 1:1000), rat anti-CD31 (BD Biosciences, 553370; dilution 1:1000), rabbit anti-Prox1 (Abcam, ab101851; dilution 1:500), and Cy3-conjugated mouse anti-SMA (MilliporeSigma, C6198; dilution 1:1000).
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