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M0761

Manufactured by Agilent Technologies
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The M0761 is a laboratory instrument designed for performing analytical measurements. It is a precise and reliable device used for a variety of applications within the scientific and research community.

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Lab products found in correlation

Ab15580, by Abcam (2 mentions) Ventana benchmark tx, by Roche (1 mentions) Lin28a, by Cell Signaling Technology (1 mentions) Map2c, by Merck Group (1 mentions) M4403, by Merck Group (1 mentions) Ab79351, by Abcam (1 mentions) Ab92547, by Abcam (1 mentions) Af835, by R&D Systems (1 mentions) Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions) Mab377, by Merck Group (1 mentions) Ab92381, by Abcam (1 mentions) Eclipse 80i microscope, by Nikon (1 mentions) Mab377, by Agilent Technologies (1 mentions) A11001, by Abcam (1 mentions) Ab6718, by Abcam (1 mentions) Ab109014, by Abcam (1 mentions) Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions) Epr1942, by Abcam (1 mentions) A5441, by Merck Group (1 mentions) Ecl detection system, by Cytiva (1 mentions)

10 protocols using m0761

1

Comprehensive Immunohistochemical Analysis of Neural Markers

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Tissue samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and then sectioned at 2 µm according to standard laboratory protocols. Immunohistochemical staining was performed on an automated staining machine (Ventana BenchMark TX, Roche Diagnostics, Mannheim, Germany). The following primary antibodies were used: Calretinin (610908, BD Biosciences, 1:1000), Caspase 3 (AF835, R&D Systems, 1:300), GFAP (M0761, Dako 1:200), Ki67 (ab15580, Abcam, 1:100), LIN28A (3978, Cell Signaling Technology, 1:100), MAP2C (M4403, Sigma-Aldrich, 1:3000), Nestin (611658, BD Biosciences, 1:200), OLIG2 (A9610, Millipore, 1:200), OTX2 (MA5-15854, Invitrogen, 1:2000), pHH3 (9706L, Cell Signaling Tech, 1:200), SOX2 (ab79351, Abcam, 1:200), Vimentin (ab92547, Abcam, 1:200), RFP (ABIN129578, Antibodies online, 1:50). Detection was performed with secondary antibodies and diaminobenzidine (DAB) as a chromogen.
Dottermusch M., Sumisławski P., Krevet J., Middelkamp M., Voß H., Siebels B., Bartsch H., Sotlar K., Meyer P., Frank S., Korshunov A., Glatzel M., Schüller U, & Neumann J.E. (2021). Co-activation of Sonic hedgehog and Wnt signaling in murine retinal precursor cells drives ocular lesions with features of intraocular medulloepithelioma. Oncogenesis, 10(11), 78.
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2

Immunohistochemical Quantification of Cerebrovascular and Neural Markers

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After nonspecific protein binding blocking, slices were incubated (1/100, 2 hours, 4°C) with the primary antibody (eNOS: 610297, BD Pharmingen, http://www.bdbiosciences.com; NeuN: MAB377, Millipore, http://www.merckmillipore.com; glial fibrillary acidic protein, GFAP: M0761, Dako, http://www.agilent.com) and were incubated secondary antibodies. Cerebral vessel density (eNOS), neuronal survival (NeuN), and astrogliosis (GFAP) are expressed as ipsilateral‐to‐contralateral (i/c) ratio.
Grandvuillemin I., Garrigue P., Ramdani A., Boubred F., Simeoni U., Dignat‐George F., Sabatier F, & Guillet B. (2017). Long‐Term Recovery After Endothelial Colony‐Forming Cells or Human Umbilical Cord Blood Cells Administration in a Rat Model of Neonatal Hypoxic‐Ischemic Encephalopathy. Stem Cells Translational Medicine, 6(11), 1987-1996.
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3

Immunostaining of FFPE Brain Samples

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Immunostainings on FFPE tissue samples were performed as previously described9 (link). In short, brain samples from three PSP patients and three NCs containing both white and grey matter were excised and fixed in formalin for min. 48 h in 10% buffered formalin before embedding in paraffin on a Leica ASP300 S tissue processor (Leica, DEU). Samples were cut on a sliding microtome into 5 µm sections. All antibodies were tested alone using HRP and 3,3′-diaminobenzidine tetrahydochloride hydrate as previously described9 (link). For immunofluorescent double labelling, slides were deparaffinized before antigen retrieval at pH 6 (NeuN) and/or 9 (IL-2, GFAP) followed by incubation with primary antibodies: Monoclonal rabbit anti-human IL-2 (1:250; Abcam; #ab92381), and monoclonal mouse anti-human NeuN (1:500; Merck Millipore; #MAB377) or monoclonal mouse anti-human GFAP (1:200; Dako; #M0761). Secondary antibodies were goat anti-mouse IgG Alexa Fluor 488 (1:200; Invitrogen; #A11001) and goat anti-rabbit IgG TRITC (1:1000; Abcam; #ab6718). Cover slides were mounted with medium containing DAPI. Stainings were investigated using a Nikon Eclipse 80i microscope. Specificity of the IL-2 antibody was tested on tonsils as a positive control (data not shown). Additionally, appropriate isotype controls were included to verify specificity.
Rydbirk R., Elfving B., Folke J., Pakkenberg B., Winge K., Brudek T, & Aznar S. (2019). Increased prefrontal cortex interleukin-2 protein levels and shift in the peripheral T cell population in progressive supranuclear palsy patients. Scientific Reports, 9, 7781.
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4

GFAP and Ki-67 Double Immunohistochemistry

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Haematoxylin and eosin and single antibody staining (GFAP, Ki-67) was done by the clinical pathology laboratory at the Massachusetts General Hospital per routine protocol. For GFAP and Ki-67 double immunohistochemistry, paraffin-embedded sections were mounted on glass slides, deparaffinized in xylene, treated with 0.5% peroxide in methanol, and rehydrated. Antigen retrieval was done using sodium citrate-based, heat-induced antigen retrieval at pH 6.0. The Dako EnVision G/2 double stain system was used for blocking, staining, and development using rabbit anti-Ki67 antibody (Abcam ab15580 at 1:300) and mouse anti-GFAP antibody (Dako M0761 at 1:100).
Tirosh I., Venteicher A.S., Hebert C., Escalante L.E., Patel A.P., Yizhak K., Fisher J.M., Rodman C., Mount C., Filbin M.G., Neftel C., Desai N., Nyman J., Izar B., Luo C.C., Francis J.M., Patel A.A., Onozato M.L., Riggi N., Livak K.J., Gennert D., Satija R., Nahed B.V., Curry W.T., Martuza R.L., Mylvaganam R., Iafrate A.J., Frosch M.P., Golub T.R., Rivera M.N., Getz G., Rozenblatt-Rosen O., Cahill D.P., Monje M., Bernstein B.E., Louis D.N., Regev A, & Suvà M.L. (2016). Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma. Nature, 539(7628), 309-313.
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5

Immunofluorescence Assay for GFAP, GSN, and F-actin

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Immunofluorescence assay was carried out following a previously published protocol.10 Anti‐GFAP Ab (1:2000, M0761; Dako), rabbit monoclonal anti‐GSN Ab (1:3000 dilution; EPR1942, ab109014; Abcam), and Alexa Fluor 568 phalloidin (1:1000 dilution; Thermo Fisher Scientific) were used.
Zhang J., Furuta T., Sabit H., Tamai S., Jiapaer S., Dong Y., Kinoshita M., Uchida Y., Ohtsuki S., Terasaki T., Zhao S, & Nakada M. (2020). Gelsolin inhibits malignant phenotype of glioblastoma and is regulated by miR‐654‐5p and miR‐450b‐5p. Cancer Science, 111(7), 2413-2422.
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6

Protein Extraction and Western Blot Analysis

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Cells were collected and proteins extracted using Lysis Buffer - 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.02% NaN3, 0.1 SDS, 1% NP40, 1 mM EDTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 1 x Protease Inhibitor Cocktail (Roche Diagnostics, IN, United States). Equal amounts of protein extracts (50 μg) were loaded onto a 10% SDS-polyacrylamide gel and resolved by standard SDS-PAGE. Proteins were then electrophoretically transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk in PBST for 60 min and tested overnight with specific antibodies at dilution 1:1000 against, mouse monoclonal glial fibrillary acidic protein (GFAP; M0761; Dako, Denmark); mouse monoclonal Vimentin (Abcam, ab8978) and mouse monoclonal ß-actin at dilution 1:5000 (A5441; Sigma Chemical, MO, United States) which was used as loading control. Subsequently, membranes were incubated with rabbit anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000) (Sigma Chemical, St. Louis, MO, United States). Blots were visualized by the ECL detection system (Amersham, United Kingdom). Results were quantified by densitometry using ImageJ Software.
Rodriguez-Jimenez F.J., Clemente E., Moreno-Manzano V, & Erceg S. (2019). Organized Neurogenic-Niche-Like Pinwheel Structures Discovered in Spinal Cord Tissue-Derived Neurospheres. Frontiers in Cell and Developmental Biology, 7, 334.
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7

Immunohistochemical Analysis of Nervous System

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Representative blocks were selected through the review of all hematoxylin and eosin slides. All immunohistochemistry staining was performed on 4 μm sections of representative blocks using a Benchmark XT stainer (Ventana/Roche Tissue Diagnostics, Indianapolis, IN, USA) according to the manufacturers' guidelines. Primary antibodies were antiglial fibrillary acidic protein (GFAP) (M0761, DAKO, 1:200), anti‐CD45 (LCA) (M0701, DAKO, 1:600), anti‐CD3 (790‐4341, VENTANA, RTU), anti‐CD20 (M0755, DAKO, 1:500), anti‐CD4 (790‐4423, VENTANA, RTU), anti‐CD8 (790‐4460, VENTANA, RTU), anti‐Bcl6 (Bcl‐6‐564, Novocastra, 1:80), anti‐CD10 (PA0270, Novocastra, RTU), anti‐CD21 (NCL‐L‐CD21‐2G9, Novocastra, 1:50), anti‐FOXP3 (ab20034, Abcam, 1:200), and anti‐NR1 (ab193310, Abcam, 1:200).
The germinal center formation was determined by integrating findings of CD20 as a B‐cell marker, Bcl6 and CD10 as germinal center markers, and CD21 as a follicular dendritic cell marker. The ratio of GFAP‐positive area, CD45‐positive immune cell count, NR1, CD3, CD20, CD4, and CD8‐positive cell numbers and each ratio were analyzed through digital scanning using Aperio GT450 slide scanner (Leica Biosystems, Buffalo Grove, IL, USA) and measurement using QuPath software (QuPath version 0.3.2).
Jang Y., Lee K., Lee C., Chu K., Lee S.K., Won J, & Lee S. (2023). Teratoma pathology and genomics in anti‐NMDA receptor encephalitis. Annals of Clinical and Translational Neurology, 11(1), 225-234.
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8

Immunohistochemical Analysis of Neural Markers

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The following antibodies have been used for immunohistochemical and immunofluorescence procedures: (1) an anti-phospho-S6 ribosomal protein (Ser235/236) rabbit polyclonal antibody (Cat. No. 2211, 1:200, Cell Signaling, Boston, MA) (2) an anti-FMRP rabbit polyclonal antibody (Ab17722, 1:5000, Abcam, Cambridge, MA) (3) an anti-GFAP mouse monoclonal antibody (M0761, prediluted, Dako, An Agilent Technologies, Carpinteria, CA) (4) an anti-OLIG2 mouse monoclonal antibody (MABN50, 1:100, EMD Millipore, Billerica, MA) and (5) an anti-mGluR5 mouse monoclonal antibody (MABN540, 1:100, EMD Millipore, Billerica, MA).
Lechpammer M., Wintermark P., Merry K.M., Jackson M.C., Jantzie L.L, & Jensen F.E. (2015). Dysregulation of FMRP/mTOR signaling cascade in hypoxic-ischemic injury of premature human brain. Journal of child neurology, 31(4), 426-432.
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9

Immunohistochemical Analysis of GFAP in Tissue

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Immunohistochemistry on formalin-fixed and paraffin-embedded tissue was performed following a previously published protocol [31 (link)]. In brief, sections at a thickness of 5 µm were deparaffinized. After microwave antigen retrieval using citrate buffer, sections were stained using the REAL EnVision Detection System peroxidase/DAB+, Rabbit/Mouse (Dako, # K5007) according to the manufacturer’s protocol. The kit uses 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a chromogen. Finally, sections were counterstained using hematoxylin. Primary antibodies and staining conditions were as follows: antigen retrieval using citrate buffer (0.1 M) for 30 min followed by anti-GFAP (Agilent Cat# M0761, RRID:AB_2109952, 1:100) incubation overnight. As positive control, an intracerebral metastasis of a breast carcinoma with an adjacent reactive border zone was used. Analyzed areas of each region is given in Additional file 1: Table S2.
Schneider Y., Gauer C., Andert M., Hoffmann A., Riemenschneider M.J., Krebs W., Chalmers N., Lötzsch C., Naumann U.J., Xiang W., Rothhammer V., Beckervordersandforth R., Schlachetzki J.C, & Winkler J. (2024). Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy. Acta Neuropathologica Communications, 12, 1.
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10

Immunohistochemistry of Cellular Markers

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IHC was performed using a Vectastain Elite kit (Vector Laboratories, Burlingame, CA) as described previously.[15, 30] Primary antibodies included the human‐specific mitochondria monoclonal antibody (1:50) (MAB1273MI, fisher scientific) and mouse anti‐glial fibrillary acidic protein (GFAP) (1:200) (M0761, AGILENT TECHNOLOGIES INC), VIM (1:200) (M0725, Dako North America,), MAP‐2 (1:200) (AB7756, Abcam Inc), Ki67 (1:20) (ab833‐500, Abcam Inc), and rabbit anti‐Nestin (NES) (1:500) (ABD69, EMD Milipore). After slides were incubated with primary antibodies for 90 min at room temperature, the appropriate biotinylated secondary antibodies (1:200) were applied and incubated for 30 min. The final signal was developed using the 3,3’‐diaminobenzidine substrate kit for peroxidase. IHC staining was assessed by combining the intensity and extent of immunopositivity.[15, 30]
Huang Y., Qi L., Kogiso M., Du Y., Braun F.K., Zhang H., Huang L.F., Xiao S., Teo W., Lindsay H., Zhao S., Baxter P., Su J.M., Adesina A., Yang J., Brabetz S., Kool M., Pfister S.M., Chintagumpala M., Perlaky L., Wang Z., Zhou Y., Man T, & Li X. (2021). Spatial Dissection of Invasive Front from Tumor Mass Enables Discovery of Novel microRNA Drivers of Glioblastoma Invasion. Advanced Science, 8(23), 2101923.
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