fluorescent-conjugates

Iizuka K, Yokomizo T, Watanabe N, Tanaka Y, Osato M, Takaku T, Komatsu N (2016) Lack of phenotypical and morphological evidences of endothelial to hematopoietic transition in the murine embryonic head during hematopoietic stem cell emergence. PLoS One 11:e0156427. doi: 10.1371/journal.pone.0156427

Summary: Hemogenic endothelial cells have been observed in several embryonic tissues, such as the dorsal aorta, the placenta and the yolk sac. Recent work also suggest that the mouse embryonic head also produces hematopoietic stem cells (HSCs)/progenitors. However, a histological basis for HSC generation in the head hasn’t been determined because the hematopoietic cluster and hemogenic endothelium have not been well characterized. The authors in this study used whole-mount immunostaining and 3D confocal reconstruction techniques to analyze both c-Kit hematopoietic cluster and Runx1 hemogenic endothelium in the whole-head vasculature. Alexa488 labeled anti-NGFr (Cat. #FL-N01AP) was used in flow cytometry. The number of c-Kit hematopoietic cells was 20-fold less in the head arteries than in the dorsal aorta. In addition, nascent hematopoietic cells, observed by a budding structure and a Runx1 hemogenic endothelium, were not observed in the head. These results suggest that head HSCs may not be or are rarely generated from the endothelium in the same manner as aortic HSCs.

Related Products: NGFr (mu p75) Rabbit Polyclonal, Alexa488-labeled affinity-purified (Cat. #FL-N01AP)

Ovsepian SV, Antyborzec I, O’Leary VB, Zaborszky L, Herms J, Oliver Dolly J (2013) Neurotrophin receptor p75 mediates the uptake of the amyloid beta (Abeta) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons. Brain Struct Funct 219(5):1527-1541. doi: 10.1007/s00429-013-0583-x

Summary: Accumulation of β-amyloid in the brain is considered one of the main causes of Alzheimer’s disease. The increase in β-amyloid is accompanied by a reduction in levels of the high affinity nerve growth factor receptor (trkA) and cognitive impairment. The authors looked at levels of the low affinity nerve growth factor receptor (p75) that do not decline. Using a 0.8-μg injection of 192-Cy3 (Cat. #FL-01) into the medial prefrontal cortex of rats the authors assessed the transport of p75 and β-amyloid by microscopy. The results indicate that the primary destinations of both p75 and β-amyloid were the late endosome and lysosome.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Li Q, Syrovets T, Simmet T, Ding J, Xu J, Chen W, Zhu D, Gao P (2013) Plasmin induces intercellular adhesion molecule 1 expression in human endothelial cells via nuclear factor-κB/mitogen-activated protein kinases-dependent pathways. Exp Biol Med (Maywood) 238(2):176-186. doi: 10.1177/1535370212473700

Summary: Intracellular adhesion molecule 1 (ICAM-1) mediates inflammatory cell migration – an early step in atherosclerosis. The authors investigated an inflammatory cascade activated by plasmin using a variety of methods, including flow cytometry with anti-mouse IgG-FITC (Cat. #FL-07) and anti-rabbit IgG-FITC (Cat. #FL-04).

Related Products: Goat Anti-Rabbit IgG, FITC-labeled (Cat. #FL-04), Goat Anti-Mouse IgG, FITC-labeled (Cat. #FL-07)

Ovsepian SV, Dolly JO, Zaborszky L (2012) Intrinsic voltage dynamics govern the diversity of spontaneous firing profiles in basal forebrain noncholinergic neurons. J Neurophysiol 108(2):406-418. doi: 10.1152/jn.00642.2011

Summary: The voltage modulation functions of the basal forebrain have commonly been associated with cholinergic cells. More recent work has suggested that noncholinergic cells have an influence on this type of neuronal activity. The authors labeled cholinergic neurons by injecting 0.8-1.6 μg of Cy3-192-IgG (Cat. #FL-01) into the lateral ventricles of rats. Patch-clamp recordings were taken from brain slices of these animals under various blockade conditions. The results demonstrate that neuropeptide Y receptors as well as ions such as Ca2+ and K+ are important for regenerative firing in basal forebrain cholinergic neurons.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Hawryluk JM, Ferrari LL, Keating SA, Arrigoni E (2012) Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons. J Neurophysiol 107(10):2769-2781. doi: 10.1152/jn.00528.2011

Summary: Using patch-clamp recordings from mouse brain slices the authors demonstrate that adenosine not only directly inhibits cholinergic neurons in the basal forebrain, it also reduces excitatory inputs to these neurons as well. Cy3-anti-mu p75 (Cat. #FL-05, 40-60 ng) was injected into the lateral cerebroventricle.

Related Products: NGFr (mu p75) Rabbit Polyclonal, Cy3-labeled affinity-purified (Cat. #FL-05)

Q: I plan to use your Secondary Antibody Conjugates, Rab-ZAP (Cat. #IT-05), and Fab-ZAP Rabbit (Cat. #IT-57) with my primary antibody and would like to observe eliminated cells using a fluorescence microscope. The idea is to co-culture cancer cells and fibroblast cells, and kill fibroblast cells only with a specific primary antibody. Then I want to observe the eliminated fibroblast cells and take pictures with a fluorescence microscope. Can you recommend a protocol?

A: In order to stain and visualize the cells that are being eliminated, it would be best to stain for Saporin using a fluorescently-tagged antibody such as the FITC-labeled Saporin antibody (Cat. #FL-02). By washing off the media after a day, and then staining for saporin, one would illuminate only the cells that have internalized the saporin (marking them for death). The cells that do not stain for saporin will live.

Related Product: FITC-labeled Saporin antibody (Cat. #FL-02)

Hess PR, Buntzman AS, Murray SL, Young EF, Frelinger JA (2009) Featured Article: Selective deletion of CD8+ T cells by saporin-coupled MHC class I tetramers. Targeting Trends 10(1)

Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin Goat Polyclonal, affinity-purified FITC-labeled (Cat. #FL-02)

Read the featured article in Targeting Trends.

See Also:

• Hess PR et al. Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood 109:3300-3307, 2007.

Yu S, Xie H, Datta A, Naidu N, Gu XX (2008) Galactose residues on the lipooligosaccharide (LOS) of moraxella catarrhalis 26404 (serotype c) form the epitope recognized by the bactericidal antiserum from conjugate vaccination. Infect Immun 76(9):4251-4258. doi: 10.1128/IAI.01570-07

Related Products: Goat Anti-Rabbit IgG, FITC-labeled (Cat. #FL-04)

Huh CY, Danik M, Manseau F, Trudeau LE, Williams S (2008) Chronic exposure to nerve growth factor increases acetylcholine and glutamate release from cholinergic neurons of the rat medial septum and diagonal band of Broca via mechanisms mediated by p75NTR. J Neurosci 28(6):1404-1409. doi: 10.1523/JNEUROSCI.4851-07.2008

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Chin JH, Ma L, MacTavish D, Jhamandas JH (2007) Amyloid beta protein modulates glutamate-mediated neurotransmission in the rat basal forebrain: involvement of presynaptic neuronal nicotinic acetylcholine and metabotropic glutamate receptors. J Neurosci 27:9262-9269. doi: 10.1523/JNEUROSCI.1843-07.2007

Summary: This work focused on the effect of amyloid beta on glutamate-mediated neurotransmission in the diagonal band of Broca. Using neurons identified by staining with Cy3-labeled 192-IgG (Cat. #FL-01, 5 µl of 1:1 diluted antibody injected into the left and right ventricle) the authors monitored the response to amyloid beta by measuring excitatory postsynaptic currents via whole-cell patch-clamp recordings. The results suggest that glutamate neurotransmission might be vulnerable to Alzheimer’s disease, and may also be a therapeutic target.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Q: I have a few questions about the Alexa488-labeled affinity-purified NGFr antibody (Cat. #FL-03). Is it specific to extracellular p75? Can you use it on live cells? Does it work on fixed cells? Does it cause activation of the p75 receptor (i.e., result in apoptosis or changes in axon outgrowth in neuronal cells)?

A: This product does recognize extracellular p75 in both live and fixed cells. As for the activation, that’s an interesting question. There is no evidence of 192-IgG either causing apoptosis or neurite outgrowth as far as I can see. Chandler et al. (1984) report that the antibody “partially inhibits the regeneration of neurites from primed PC12 cells,” and it enhances NGF binding. But that’s about it, despite several studies being done with PC12 cells and in vivo. We assume all this holds upon treatment with 192-IgG-SAP (Cat. #IT-01) — until the cell dies from saporin poisoning.

Chandler CE, Parsons LM, Hosang M, Shooter EM (1984) A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells. J Biol Chem 259(11):6882-6889.

Related: Alexa488-labeled affinity-purified NGFr antibody (Cat. #FL-03)

Momiyama T, Fukazawa Y (2007) D1-like dopamine receptors selectively block P/Q-type calcium channels to reduce glutamate release onto cholinergic basal forebrain neurones of immature rats. J Physiol 580(Pt 1):103-117. doi: 10.1113/jphysiol.2006.125724

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Momiyama T, Zaborszky L (2006) Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons. J Neurophysiol 96(2):686-694. doi: 10.1152/jn.00507.2005

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Xu C, Michelsen KA, Wu M, Morozova E, Panula P, Alreja M (2004) Histamine innervation and activation of septohippocampal GABAergic neurones: Involvement of local ACh release. J Physiol 561(Pt 3):657-670. doi: 10.1113/jphysiol.2004.071712

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Wu M, Newton SS, Atkins JB, Xu C, Duman RS, Alreja M (2003) Acetylcholinesterase inhibitors activate septohippocampal GABAergic neurons via muscarinic but not nicotinic receptors. J Pharmacol Exp Ther 307(2):535-543. doi: 10.1124/jpet.103.052514

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Wu M, Xu C, Alreja M (2002) Physiological and pharmacological characteristics of the inhibitory muscarinic response in septohippocampal cholinergic neurons. Neuroscience 2002 Abstracts 35.7. Society for Neuroscience, Orlando, FL.

Summary: Septohippocampal cholinergic neurons in the MSDB provide the hippocampus with almost its entire ACh and also release ACh locally within the MSDB. The released ACh sustains activity in the GABAergic limb of the septohippocampal pathway. Septohippocampal cholinergic neurons undergo atrophy in neurodegenerative disorders associated with loss of cognition. In a recent study we demonstrated that 65% of septohippocampal cholinergic neurons are inhibited by ACh via muscarinic receptors. Because of the importance of ACh and septohippocampal cholinergic neurons in cognition, we studied the physiological and pharmacological properties of the muscarinic response in MSDB neurons. Using intracellular and whole-cell recordings, we tested the effects of muscarine on retrogradely-labeled septohippocampal cholinergic neurons in vitro in rat brain slices. The cells were labeled using the Cy3-192IgG, a selective marker of septohippocampal cholinergic neurons. Prolonged (10-15 mins) but not short (1-2 min) applications of muscarine or oxotremorine produced a marked desensitization (>50%). The muscarine-induced outward current was found to be mediated via direct as well as indirect mechanisms. It reversed at Ek and was blocked by external barium. The M2/M4 antagonist, methoctramine blocked the muscarine response in only 10% of the neurons tested and tropicamide, an M4-prefering antagonist, blocked the muscarine response in 5/5 neurons tested, suggesting possible involvement of M4 receptors.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Harikumar KG, Pinon DI, Wessels WS, Prendergast FG, Miller LJ (2002) Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor. J Biol Chem 277(21):18552-18560. doi: 10.1074/jbc.M201164200

Related Products: 192-IgG Mouse Monoclonal, Alexa488-labeled (Cat. #FL-03)

Peterson WE, Jordan LM, Brownstone RM (2001) Immunolesioning of identified motoneuron pools following intramuscular injection of the immunotoxin, 192-IgG-saporin, in neonatal rats. Neuroscience 2001 Abstracts 626.14. Society for Neuroscience, San Diego, CA.

Summary: Studies have shown that the immunotoxin, 192-IgG-Saporin, can selectively lesion p75-positive cholinergic neurons of the basal forebrain in adult rats. Here we demonstrate the novel use of 192-IgG-Saporin to induce MN loss following intramuscular (I.M.) injection in neonatal rats. Two days following I.M. injection of 192-IgG-Cy3, neonatal rats (but not adult rats or neonatal mice) had Cy3-labeled MNs. This suggests that the 192-IgG antibody and its conjugates can be internalised by receptor-mediated endocytosis and retrogradely transported to spinal motor neurons. To induce MN loss, the left hind limb musculature of anaesthetised Sprague-Dawley rats were exposed, and several muscles injected with 0.5μg of 192-IgG-Saporin (Chemicon). Right hind-limb muscles were injected with DiI. Animals were sacrificed 25 days later. Ten μm coronal sections were obtained using a cryostat and Nissl stained. The neonatal rats showed signs of a locomotor deficit 2.5 weeks post injection with 192-IgG-Saporin, which increased slightly in severity over the next week and a half. Nissl stained coronal sections of the lumbar region showed an obvious MN deficit on the 192-IgG-Saporin treated side compared to control side. The injected muscles were also severely atrophic, a not unexpected finding given that they too express p75 receptors. We conclude that 192-IgG-Saporin can be used to lesion MN pools when IM injected in neonatal rats. This model may prove useful for testing cell replacement therapies for the treatment of MN diseases like amyotrophic lateral sclerosis (ALS).

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Camara AL, Pereira EF, Alkondon M, Randall WR, Castro NG, Cintra WM, Albuquerque EX (2001) Septal innervation of the hippocampus regulates expression α7 nicotinic receptors in CA1 and CA3 pyramidal neurons. Neuroscience 2001 Abstracts 145.1. Society for Neuroscience, San Diego, CA.

Summary: To investigate the effects of septal innervation on expression of α7 nicotinic receptors (nAChRs) in CA1 and CA3 pyramidal neurons in the hippocampus, the patch-clamp technique and confocal microscopy were applied to organotypic hippocampal cultures and septal-hippocampal co-cultures. In the co-cultures, septal fibers labeled with DiI were visualized in the hippocampus. Field stimulation of septal fibers also resulted in postsynaptic currents that could be recorded from CA1 and CA3 pyramidal neurons in the hippocampus. These currents had glutamatergic, GABAergic and cholinergic components. The latter originated most likely from the septal cholinergic neurons that were labeled in situ with the cholinergic marker Cy3-192 IgG. α7 nAChRs in the somatodendritic region of CA1 and CA3 pyramidal neurons in the hippocampus in cultures and co-cultures were activated by the α7 nAChR agonist choline, which elicited type IA currents, and were visualized by labeling with rhodamine-conjugated α-bungarotoxin (Rho-α-BGT). After 21 days in vitro, the amplitude of type IA currents was substantially smaller in pyramidal neurons in septal-hippocampal co-cultures than in hippocampal oragnotypic cultures. Labeling of the somatodendritic region of hippocampal pyramidal neurons with Rho-α-BGT was also less intense in the organotypic co-cultures than in cultures. These results suggest that functional septal innervation of the hippocampus regulates the expression of α7 nAChRs in hippocampal pyramidal neurons.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #FL-01)

Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat. J Neurosci 21(18):7273-7283. doi: 10.1523/JNEUROSCI.21-18-07273.2001

Summary: Orexin (also knows as hypocretin) peptides are produced exclusively by neurons in the lateral hypothalamus, however non-specific lesioning in this region has not produced narcoleptic-like sleep. Gerashchenko et al. use orexin-SAP (490 ng/0.5 µl; Cat. #IT-20) to specifically eliminate orexin neurons in rats. The treated rats displayed several sleep disturbances found in narcolepsy, including increased slow-wave sleep, and sleep-onset REM sleep periods. The data suggest that orexin-SAP can be used to create a model for narcolepsy in rats.

Related Products: Orexin-B-SAP (Cat. #IT-20), Saporin Goat Polyclonal, affinity-purified FITC-labeled (Cat. #FL-02), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP)