Saturday, November 22, 2008

Excellent TRPV1-N IHC and WB

Kudos to Dr. Federica MF van Dissel-Emiliani and her team for the excellent Immunohistochemistry and Western Blot results using our TRPV1-N Antibody(Catalog #: RA10110) . The antibody was in their study demonstrating the sensitivity of spermatogenesis to capsaicin.

Here's the related publication:

Sefika C Mizrak, Bart M Gadella, Hatice Erdost, Aytekin Ozer, Ana MM van Pelt, Federica MF van Dissel-Emiliani. Spermatogonial stem cell sensitivity to capsaicin: An in vitro study. Reproductive Biology and Endocrinology 2008, 6:52 doi:10.1186/1477-7827-6-52.
Anti TRPV1 antibody staining: Bouin's fixed, paraffin embedded 5 um-thick rat testis sections were deparaffinized and boiled in a microwave oven (700 Watt) 3x10 min in sodium citrate buffer (0.1 mM, pH=6) for antigen retrieval. All subsequent incubations were performed for 1 hour at room temperature. The slides were then blocked with 5 % goat serum in 1 % BSA/PBS and incubated with the rabbit anti human - VR1 antibody (Neuromics, Edina, MN, USA; 1:500 in 1% BSA/PBS). Biotinilated goat anti-rabbit secondary antibody (BA-1000, Vector Labs; 1:200 in 1% BSA/PBS) was then applied. The ABC kit was finally used according to the manufacturer's instructions. Antibody reactivity was finally detected by diaminobenzidine staining (DAB, Sigma, St. Louis, MO, USA). Sections were counterstained with hematoxylin, dehydrated, mounted with Pertex and studied. Goat serum was applied on control sections.
Image: Photomicrograph of a section through an adult rat testis showing TRPV1 labelling of premeiotic germ cells, at stage II of the seminiferous epithelium. Arrow, undifferentiated spermatogonia; arrow head, early pachytene spermatocytes; asterisk, Sertoli cells.
SDS-PAGE and Western blotting: Protein lysates from the cell lines Gc-5spg and Gc-6spg and the control glioma cell line (A10-85) were prepared in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) including 1 mM phenylmethylsulfonylfluoride. Of each sample, 50 μg were separated on a 12% SDS-polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). Western blots were blocked using Blotto-A, containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (10 mM Tris; 150 mM NaCl, pH 7.6), including 0.05% Tween-20. Rabbit polyclonal anti-VR1 antibody (Neuromics) was diluted 1:1000 in Blotto-A and incubated for 1 h at room temperature. Blots were washed with Tris-buffered saline with 0.05% Tween-20. After incubation with goat anti-rabbit-HRP (P-0260 Dako Cytomation, 1:5000 inBlotto-A) secondary antibody for 1 h, blots were incubated with the electrochemiluminescence kit (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK)and exposed to an x-ray film (RX-omat, Kodak, Chalone / Saone, France).

Wednesday, November 12, 2008

DRG Neurons Now Available.

Primary Rat DRGs are live neurons isolated from micro-surgically dissected regions of day 18 embryonic Sprague/Dawley rat brain. These cells are prepared fresh each week and shipped in a nutrient rich medium that keeps the cells alive for up to 14 days under refrigeration.
Please note: It is important to review Protocol/Datasheet prior to ordering. There is a unique step for making the dissociation enzyme solution. Do not hesitate to call or e-mail me (612-801-1007 or should you have questions.

Image: DRGs cultured on Calf Skin Collagen.

-A dissection and Tissue Culture Manual of the Nervous System (1989). A. Shahar, J.D. Vellis, A. Vernadakis, B. Haber (Eds.), Dissociated Spinal Cord - Dorsal Root Ganglion Cultures on Plastic Tissue Culture Dishes and Glass Coverslips and Wells (pp.219-222). Wiley-Liss, Inc. J.L. Werth, -S.A. Thayer (1994) Mitrochondria Buffer Physiological Calcium Loads in Cultured Rat Dorsal Root Ganglion Neurons, The Journal of Neuroscience, 14(1), 348-356

Monday, November 03, 2008

Power Trio of Pain Research Antibodies

I received a call from a customer asking if there were any publications referencing our rabbit Alpha 2a Adrenergic Receptor antibody. The resulting search found an article referencing excellent results using a trio of of our pain research antibodies. The research was conducted by our friend, Dr. Hui-Lin Pan and his team at University of Texas M.D. Anderson Cancer Center.

Shao-Rui Chen, Hao-Min Pan, Timothy E. Richardson, and Hui-Lin Pan. Potentiation of Spinal α2-Adrenoceptor Analgesia in Rats Deficient in TRPV1- Expressing Afferent Neurons. Published online 2007 March 24. doi: 10.1016/j.neuropharm.2007.03.009.

Images: Double immunofluorescence labeling showing α2C-AR- and TRPV1-immmunoreactivity in the spinal cord dorsal horn of one vehicle-treated and one RTX-treated rat. Representative confocal images showing α2C-AR- and VR1 C-terminus (TRPV1)-immunoreactivities in the spinal dorsal horn of one vehicle- and one RTX-treated rat. All images are single confocal optical sections. Scale bar, 100 μm. Inset: high-magnification images (scale bar = 10 μm) showing the distribution of α2C-AR- and TRPV1-immunoreactivity in the lamina I and II. Neuropharmacology. 2007 June; 52(8): 1624–1630.

Rabbit VR1 N-Terminus (TRPV1) is also referenced.

Related Reagents:
Alpha 2c
Pain and Inflammation Antibodies
Vision and Retina Antibodies