كله يشن التحليل المناعى للالجنينية الاطراف الأوعية الدموية الجلدية : نظام نموذجي لدراسة الأوعية الدموية المتفرعة التشكل في الأجنة

Biology

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Li, W., Mukouyama, Y. Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo. J. Vis. Exp. (51), e2620, doi:10.3791/2620 (2011).

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Abstract

Materials

Antibodies

Pan-endothelial cell marker

Antibody Species Company Catalogue # Working condition
PECAM-1 Armenian hamster (M) Chemicon MAB1398Z 1:100 dilution#1
PECAM-1 Rat (M) BD 553369 1:300 dilution
VEGFR2 Rat (M) eBioscience 14-5821-82 1:200 dilution
CD34 Rat (M) eBioscience 13-0341 1:300 dilution
Collagen IV Rabbit (P) AbD Serotec 2150-1470 1:300 dilution#2

Arterial endothelial cell marker

Antibody Species Company Catalogue # Working condition
Neuropilin1 Rabbit (P) The Alex Kolodkin’s lab#3 1:3000 dilution
Unc5H2 Goat (P) R&D AF1006 1:200 dilution

Venous endothelial cell marker

Antibody Species Company Catalogue # Working condition
EphB4 Goat (P) R&D AF446 1:100 dilution

Lymphatic endothelial cell marker

Antibody Species Company Catalogue # Working condition
LYVE-1#4 Rabbit (P) Abcam ab14917 1:200 dilution
LYVE-1#4 Rat (M) MBL D225-3 1:200 dilution
Prox-1 Rabbit (P) Chemicon AB5475 1:1000 dilution
Prox-1 Goat (P) R&D AF2727 1:50 dilution
Neuropilin2 Rabbit (P) Cell Signaling 3366 1;100 dilution
Podoplanin Syrian hamster (M) Hybridoma Bank 8.1.1 1:200 dilution

Smooth muscle cell/pericyte marker

Antibody Species Company Catalogue # Working condition
αSMA-Cy3 Mouse (M)#5 Sigma c-6198 1:500 dilution#6
NG2 Rabbit (P) Chemicon AB5320 1:200 dilution
SM22α Rabbit (P) Abcam ab14106 1:200 dilution

Antibodies forGFP reporter

Antibody Species Company Catalogue # Working condition
GFP Rabbit (P) Invitrogen A11122 1:300 dilution
GFP Rat (M) Nacalai tesque 04404-84 1:1000 dilution
GFP Chick (P) Chemicon P42212 1:300 dilution

Antibodies for LacZ reporter

Antibody Species Company Catalogue # Working condition
β-gal Rabbit (P) MP Biomedical 55976 1:5000 dilution
β-gal Goat (P) AbD Serotec 4600-1409 1:500 dilution
β-gal Chick (P) Abcam ab9361 1:200 dilution

Antibodies for peripheral axon

Antibody Species Company Catalogue # Working condition
2H3 Mouse (M)#7 Hybridoma Bank 2H3 1:200 dilution
Tuj1 Mouse (M)#8 Covance MMS-435P 1:500 dilution
Peripherin Rabbit (P) Chemicon AB1530 1:1000 dilution

Antibodies for migrating Schwann cells

BFABP Rabbit (P) The Thomas Müller’s lab#9 1:3000 dilution

(P): polyclonal antibody, (M): monoclonal antibody

#1: Goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch 127-165-160) antibody should be used as a secondary antibody.

#2: The Collagen IV antibody can be used to detect blood vessels after in situ hybridization.

#3: The Neuropilin1 antibody is kindly provided by the Alex Kolodkin’s lab in the Johns Hopkins University. Sheep anti-human Neuropilin1 antibody is available in R&D (AF3870), although we have not tested it yet.

#4: The LYVE-1 antibodies also detect a subset of macrophages in the embryonic skin.

#5: The anti-αSMA antibody is mouse IgG2a monoclonal antibody.

#6: The Cy3-conjugated αSMA antibody is incubated for 1 hour at room temperature together with secondary antibodies for other primary antibodies.

#7: 2H3 antibody is mouse IgG1 monoclonal antibody against neurofilament.

#8: Tuj1 antibody is mouse IgG2a monoclonal antibody against Neuron-specific class III beta-tubulin.

#9: The BFABP (brain-specific fatty acid binding protein) antibody is kindly provided by the Thomas Müller’s lab in Max-Delbrück-Center for Molecular Medicine.

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References

  1. Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell. 109, 693-705 (2002).
  2. Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback. Development. 132, 941-952 (2005).
  3. Wang, H. U., Chen, Z. F., Anderson, D. J. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 93, 741-753 (1998).
  4. Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell. 4, 403-414 (1999).

Comments

5 Comments

  1. excellent! I learned a lot

    Reply
    Posted by: Anonymous
    August 12, 2011 - 5:38 PM
  2. Very detailed and helpful !!

    Reply
    Posted by: Anonymous
    February 6, 2012 - 9:54 AM
  3. it is the first time I saw the image of nerves and vessels so lively. it is a GREAT model to study the dynamic development during mouse embryonic stage even in adult mouse! it is amazing that you found this technic!

    Reply
    Posted by: Anonymous
    February 8, 2012 - 11:26 AM
  4. Thanks for the very useful method!
    Have you tried adult mouse skin? What would you recommend to modify if I do whole mount staining of adult mouse skin?
    Thanks a lot!

    Reply
    Posted by: Dong W.
    February 6, 2013 - 10:36 PM
  5. Yes, this method works on adult mouse skin as well. The detailed protocols are descibed in our revision paper submitting to Developmental Dynamics. Thanks!

    Reply
    Posted by: wenling l.
    February 7, 2013 - 2:19 PM

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