Margarida Martins-Oliveira, Simon Akerman, Philip R. Holland, Jan R. Hoffmann, Isaura Tavares, Peter J. Goadsby. Neuroendocrine signaling modulates specific neural networks relevant to migraine. Neurobiology of Disease. Available online 17 January 2017. http://dx.doi.org/10.1016/j.nbd.2017.01.005

...Immunohistochemistry

Using the same cohort of animals of blood glucose measurements, we studied the effect of intravenous treatment with insulin (10 U·kg− 1), glucagon (100 μg·200 μl− 1) and leptin (1 mg·kg− 1) or sham control (0.2–0.3 ml sterile water) on pERK1/2 expression within the TCC and the hypothalamus. At the end of each experiment, animals were euthanized with pentobarbital sodium (60 mg/kg, i.v.; Euthatal®, 20% w/v, JML) and perfused with 250 ml of heparinized (1%) phosphate-buffered saline (PBS) through the ascending aorta, followed by 300 ml of a fixative solution containing 4% paraformaldehyde in 0.01 M phosphate buffer, pH 7.4. The brain and upper spinal cord were removed, immersed in the same fixative for 2–4 h followed by 30% sucrose in 0.01 M PBS and 0.01% sodium azide, at 4 °C, and sliced at 40 μm in coronal orientation on a cryostat. Tissue was then processed for free-floating immunohistochemistry. One in four sequential transverse brain sections containing the hypothalamus and the TCC from each rat were collected, washed, blocked and incubated with a rabbit antiserum against the phosphorylated ERK1 and ERK2 isoforms (pERK1/2; 1:1000; 48 h at 4–8 °C: Neuromics, USA). Immunodetection was achieved with a biotinylated swine anti-rabbit antiserum (1:200; 1 h; Dako, Denmark), followed by an ABC solution (1:200, 1 h; ABC Elite kit, Vector Laboratories, UK) and a colorimetric reaction with 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.05 M Tris-HCl buffer containing 0.003% hydrogen peroxide. Sections were then washed in PBS, mounted on gelatin-coated glass slides, cleared in xylene, cover-slipped with Eukitt® and analyzed by light microscopy. Histological delineation of the TCC and the hypothalamus was made using the rat brain atlas (Paxinos and Watson, 2005)....

MARIA CONCETTA OLIANAS, Simona Dedoni, and PIER LUIGI ONALI. LPA1 mediates antidepressant-induced ERK1/2 signalling and protection from oxidative stress in glial cells. Published online before print September 7, 2016, doi: 10.1124/jpet.116.236455.

...Membranes were incubated overnight at 4 °C with one of the following primary antibodies: anti-phospho-ERK1 (Thr202/Tyr204) / ERK2(Thr185/Tyr187) (RA 15002) (1:15,000) (Neuromics, Northfield, MN, USA ...

Lidia Bravo, Juan A. Mico, Raquel Rey-Brea, Carmen Camarena-Delgado, Esther Berrocoso. Effect of DSP4 and desipramine in the sensorial and affective component of neuropathic pain in rats. Progress in Neuro-Psychopharmacology and Biological Psychiatry. Available online 12 May 2016. doi:10.1016/j.pnpbp.2016.05.002

... with 0.1% Tween-20), the blots were blocked with 5% Bovine Serum Albumin (BSA; Sigma, Spain)in TBST and incubated overnight at 4 °C with a mixture of primary antibodies diluted in TBST plus 5% BSA: rabbit anti-phospho-ERK1/2 (1:5000; Neuromics (RA15002), USA ...

Simona Dedoni, Maria C. Olianas, Angela Ingianni, Pierluigi Onali. Interferon-β Inhibits Neurotrophin 3 Signalling and Pro-Survival Activity by Upregulating the Expression of Truncated TrkC-T1 Receptor. Molecular Neurobiology. pp 1-19. First online: 18 February 2016. DOI 10.1007/s12035-016-9789-2

... anti-Na+/K+- ATPase α1 subunit (sc-21712), anti-tamalin (sc-55951), anti- phospho-tyrosine(PY99) (sc-7020) (Santa Cruz Biotechnology); rabbit polyclonal anti-actin (Sigma- Aldrich);anti-phospho-ERK1 (Thr202/Tyr204) / ERK2 (Thr185/Tyr187) (Neuromics, Northfield, MN ...

Gisela Patrícia da Silva Borges , Juan Antonio Micó Segura , Fani Lourença Moreira Neto , Esther Berrocoso. Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons. DOI: http://dx.doi.org/10.1093/ijnp/pyv019 First published online: 25 February 2015

Western Blotting: The membranes were blocked with 5% Bovine Serum Albumin (BSA; Sigma, Spain) in TBST and probed overnight at 4 ºC with a rabbit anti-phospho-ERK1/2 (1:5,000; Neuromics).

Immunohistochemistry: Brains were removed and processed for free-floating immunohistochemistry. One in five sequential transverse brain sections (30 µm) containing the PVN from each rat were washed, blocked and incubated with a rabbit antiserum against the phosphorylated ERK1 and ERK2 isoforms (pERK1/2; 1:1000; 48 hours at 4-8ºC: Neuromics, USA). Immunodetection was achieved with a biotinylated donkey anti-rabbit antiserum (1:500; 1 hour; Jackson ImmunoResearch, USA), followed by an ABC solution (1:200, 1 hour; ABC Elite kit, Vector Laboratories, UK) and a colorimetric reaction with 3,3-diaminobenzidine tetrahydrochloride (DAB; 10 min) in 0.05M Tris-HCl buffer containing 0.003% hydrogen peroxide (Cruz et al., 2005). Sections were then washed in PBS, mounted on gelatin-coated glass slides, cleared in xylene, cover-slipped with DPX and analyzed by light microscopy.

Maria C. Olianas, Simona Dedoni, Pierluigi Onali. Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. Volume 1843, Issue 12, December 2014, Pages 3004–3017

...anti-phospho-ERK1 (Thr202/Tyr204)/ERK2(Thr185/Tyr187) (1:15 000) (Neuromics, Edina, MN)...

Coelho, A., Oliveira, R., Rossetto, O., Cruz, C.D., Cruz, F. and Avelino, A. (2014), Intrathecal administration of botulinum toxin type A improves urinary bladder function and reduces pain in rats with cystitis. European Journal of Pain. doi: 10.1002/ejp.513

...Rabbit anti-phospho ERK1/2 protein (RA15002) was obtained from Neuromics, Edina, MN, USA...

Borges, G.S., Berrocoso, E., Ortega-Alvaro, A., Mico, J.A. and Neto, F.L. (2013), Extracellular signal-regulated kinase activation in the chronic constriction injury model of neuropathic pain in anaesthetized rats. European Journal of Pain, 17: 35–45. doi: 10.1002/j.1532-2149.2012.00181.x

...After noxious mechanical stimulation (or not), the anaesthetized animals were transcardially perfused through the ascending aorta with 250 mL of oxygenated Tyrode's solution followed by 750 mL of paraformaldehyde 4% in phosphate buffer, 0.1 mol/L pH 7.2. Brains and spinal cords were processed for free-floating immunohistochemistry (Cruz et al., 2005; Nascimento et al., 2011) against pERK1/2. One in each five 40-μm brainstem or L4, L5 and L6 spinal cord sequential transverse sections from each rat went through washes and incubation in blocking solution (Cruz et al., 2005), and were incubated in antibody raised in rabbit against the phosphorylated isoforms ERK1 and ERK2 (pERK1/2; 1:1000; 48 h at 4–8 °C; Neuromics, Edina, Minnesota, USA). Immunodetection was achieved upon incubation in biotinylated swine anti-rabbit antiserum (1:200; 1 h; DAKO, Glostrup, Denmark), followed by ABC solution (1:200, 1 h; ABC Elite kit; Vector Laboratories, Peterborough, UK) and reaction with 3,3-diaminobenzidine tetrahydrochloride (DAB; 10 min) (Cruz et al., 2005).

To evaluate the nature of the pERK1/2 IR cells in spinal cord sections of CCI rats, double immunolabelling against pERK1/2 and the anti-neuronal nuclear antigen (NeuN), specific for mature neurons in vertebrates (Mullen et al., 1992), was performed by incubation (two overnights) in rabbit anti-pERK1/2 (1:1000) and mouse anti-NeuN (1:100; Millipore, Chemicon, CA, USA) primary antibodies. The immunoreaction was revealed using as secondary antibodies biotinylated donkey anti-rabbit (1:200) followed by incubation in Streptavidin conjugated with alexa®488 (1:200; Invitrogen, Molecular Probes, USA) and donkey anti-mouse conjugated with alexa®568 (1:1000; Invitrogen, Molecular Probes). A similar procedure was used to evaluate the neurochemical nature of noradrenergic [sheep anti-tyrosine hydroxylase (TH), 1:2000; Abcam, Cambridge, UK] or serotonergic [sheep anti-tryptophan hydroxylase (TrpOH), 1:500; Millipore, Chemicon] areas in supraspinal adjacent sections. Biotinylated donkey anti-rabbit (1:200), streptavidin conjugated with alexa®488, and donkey anti-sheep conjugated with alexa®568 (1:1000) were used. Specificity was controlled by omitting each primary antibody in various sets of immunoreactions...

John P.M. Whitea, Chin Wing Koa, Antonio Rei Fidalgoa, Mario Cibellia, Cleoper C. Paulea, Peter J. Andersona, Celia Cruzb, Szabolcs Gombad, Klara Matesze, Gabor Veressd, Antonio Avelinob and Istvan Nagya. Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons. European Journal of Pain.doi:10.1016/j.ejpain.2010.12.006.

...pERK1/2 (1:1000, Neuromics; RA15002)...

Mauricio P. Pinto, Melanie M. Badtke, Michelle L. Dudevoir, J. Chuck Harrell, Britta M. Jacobsen and Kathryn B. Horwitz. Vascular Endothelial Growth Factor Secreted by Activated Stroma Enhances Angiogenesis and Hormone-Independent Growth of Estrogen Receptor–Positive Breast Cancer.Cancer Research 70, 2655, April 1, 2010. Published Online First March 23, 2010; doi: 10.1158/0008-5472.CAN-09-4373 © 2010 American Association for Cancer Research.

...Phosphorylated extracellular signal-regulated kinase (p-ERK) was assayed by immunohistochemistry (rabbit polyclonal; Neuromics). Statistical analyses Data were analyzed with GraphPad software using either Student's t test or ANOVA followed by a Tukey's...

Pierluigi Onali, Simona Dedoni and Maria C. Olianas. Direct Agonist Activity of Tricyclic Antidepressants at Distinct Opioid Receptor Subtypes. JPET January 2010 vol. 332 no. 1 255-265 .

...rabbit polyclonal anti-phospho-ERK1/2 (pERK1/2) from Neuromics...

Taiichiro Chikama, Chia-Yang Liu, Johanna T.A. Meij, Yasuhito Hayashi, I-Jong Wang, Liu Yang, Teruo Nishida and Winston W.Y. Kao. Excess FGF-7 in Corneal Epithelium Causes Corneal Intraepithelial Neoplasia in Young Mice and Epithelium Hyperplasia in Adult Mice.  American Journal of Pathology. 2008;172:638-649.

... rabbit anti-phosphoERK1/2 (Neuromics, Inc., Edina, MN)...

Pierluigi Onali and Maria C Olianas. N-Desmethylclozapine, a Major Clozapine Metabolite, Acts as a Selective and Efficacious -Opioid Agonist at Recombinant and Native Receptors. Neuropsychopharmacology advance online publication 12 July 2006.

...The cells were then washed and incubated with a rabbit anti phospho-ERK1/2 affinity purified antibody (1 : 1000 dilution in PBS containing 0.25% Triton X-100 and 1.5% NGS; Neuromics, Edina, MN)...