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.
Mab377
Manufactured by Agilent Technologies
Sourced in Canada
The MAB377 is a piece of laboratory equipment manufactured by Agilent Technologies. It is designed to perform a specific function, but without further information, a detailed and unbiased description cannot be provided while maintaining conciseness. Additional details about the core function of this product would require more context.
Automatically generated - may contain errors
Lab products found in correlation
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab5905, by Merck Group (2 mentions)
Z033429, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
M7240, by Agilent Technologies (2 mentions)
M0761, by Agilent Technologies (1 mentions)
Ab92381, by Abcam (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
A11001, by Abcam (1 mentions)
Ab6718, by Abcam (1 mentions)
Fluoview 1000 ix81, by Olympus (1 mentions)
Lsm 510, by Olympus (1 mentions)
A11039, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
Goat anti rabbit 647, by Thermo Fisher Scientific (1 mentions)
As 55043a, by AnaSpec (1 mentions)
Nis elements ar, by Nikon (1 mentions)
A1 laser scanning confocal microscope, by Nikon (1 mentions)
Nis elements ar analysis, by Nikon (1 mentions)
4 6 diaminodino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
12 protocols using mab377
1
Immunostaining of FFPE Brain Samples
2
Immunohistochemical Analysis of Brainstem Tissues
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Slices used for patch-clamp or fresh brainstem slices (~120 µm) were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Free-floating sections were blocked in 4% goat serum and 0.3% (w/v) Triton X-100 in PBS for 1 h and then incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: mouse anti-CNPase (1:200; Sigma, C5922), mouse anti-O1 (1:500; Millipore, MAB5540), mouse anti-Nav1.2 (1:50; Neuromab, 75-024 Clone K69/3), mouse anti-PLP/DM20 (1:100; Thermo Fisher, MA180034), mouse anti-NeuN (1:200; Millipore, MAB377), rabbit anti-GFAP (1:500; DAKO, Z033429), guinea pig anti-vGluT1 (1:1000; Millipore, AB5905), rabbit anti-NG2 chondroitin sulphate proteoglycan (1:50; Santa Cruz Biotechnology, sc-20162), mouse anti-MBP (1:500; BioLegend, SMI-99P) and rabbit anti-Nav1.6 (1:200, Alamone, ASC-009). Tissues were then incubated with different Alexa-conjugated secondary antibodies (1:500; Invitrogen) for 2 h at room temperature. After five rinses with PBS, slices were coverslipped using mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vectashield; Vector Laboratories) to counterstain cell nuclei. Stained slices were viewed on a confocal laser-scanning microscope (Zeiss LSM-510 or Olympus IX81 Fluoview 1000) at 488, 568 and 633 nm using a 40× or 60× oil-immersion objective.
3
Immunohistochemistry of Mouse Brain Slices
4
Immunolabeling of Neurogenic Markers in Brain
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For the BrdU immuno-labeling, the brain sections were rinsed three times in 0.1 M PBS and incubated in 2 N HCl at 37°C for 15 min, followed by 0.1 M borate buffer (pH = 8.6) for 10 min and washed 3× with 0.1 M PBS. Sections were incubated in blocking solution for 40 min. Then, sections were incubated with some combinations of the following primary antibodies: rat anti-BrdU (1:500; AbD Serotec Cat # OBT0030, RRID:AB_609568 ), mouse anti-NeuN (1:500; Millipore Cat # MAB377, RRID:AB_2298772 ), rabbit anti-GFAP (1:100; Dako Cat # Z0334, RRID:AB_10013382 ), rabbit anti-Sox2 (1:500, Abcam Cat # AB97959; RRID:AB_2341193 ), guinea pig anti-doublecortin (DCX, 1:1000: Millipore Cat# AB2253, RRID:AB_1586992 ) in blocking solution at 4°C overnight. Sections were rinsed 3× with 0.1 M PBS, and incubated in 0.1 M PBS containing 10% fetal bovine serum and conjugated secondary antibodies (Alexa Fluor® 488 anti-rat Cat # A-21208; Alexa Fluor® 594 anti-rat Cat # A-11007; Alexa Fluor® 488 anti-mouse Cat # A32723; Alexa Fluor® 594 anti-rabbit; Alexa Fluor® 594 Cat# R37117; anti-guinea pig Cat# A-11076; dilution 1:1000; Thermo Fisher) for 1 h at room temperature and washed 3× with 0.1 M PBS. Nuclear counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI; Abcam Cat # ab104139, Cambridge, MA, USA).
5
Immunofluorescence Protocol for Brain Sections
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Immunofluorescence was performed as previously described [17 (link)]. Briefly, brains were sectioned at a thickness of 30 µm and left 1 h in blocking solution (10% normal donkey serum, 0.3% Triton-X1000, 0.04% NaN3 in PBS). Following the blocking step, the sections were incubated with primary antibodies (diluted in blocking solution) overnight at 4 °C. The primary antibodies used were rabbit anti-Ascl1 (Cosmo Bio Co., CAC-SK-T01-003, concentration 1:250), goat anti-Dcx (Santa Cruz, SC-8066, concentration 1:500), mouse anti-NeuN (Millipore, MAB377, concentration 1:500), rabbit anti-S100β (DAKO, Z0311, concentration 1:200, antibody was directly conjugated with fluorophore Alexa-647) and rat anti-Ki67 (eBioscience, 14-5698, concentration 1:1000). Subsequently, the sections were washed three times for 5 min in PBS before incubation with secondary antibodies (1:500 in blocking solution) against the species for the specific primary antibodies and conjugated with the fluorophores Alexa-488, Cy3 and Alexa-647. For Ascl1 detection we used a Tyramide Signal Amplification (TSA) protocol (PerkinElmer, NEL701001KT).
6
Comparative AAV Serotype Tropism
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To compare the tropism and spread of three different AAV serotypes, a CMV-GFP construct (hCMV-chI-EGFP-WPRE-SV40p(A)) was used to produce AAV2/5, AAV2/8, and AAV2/9 virus as described above with titers of 1.4 × 1013 vector genomes (vg)/mL, 1.3 × 1013 vg/mL, 5.2 × 1012 vg/mL, respectively. ICV injections of 2 × 1010 vg of virus per animal were performed as described in the section “ICV injections” in the “materials and methods” section. Brains were collected at 5 or 8 weeks, or 9 months of age; spinal cords were additionally collected at 9 months of age. Immunohistochemistry was performed after tissue processing using NeuN (1:500, Millipore, MAB377), GFAP (1:1,000, DAKO, Z0334), and Olig2 (1:1,000, Millipore, AB9610) to determine viral tropism.
7
Comprehensive Histological Evaluation of Tissues
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Standard histological preparations, 4 μm formalin-fixed paraffin-embedded (FFPE) sections stained with hematoxylin & eosin were supplemented with immunohistochemical preparations. Antibodies to the following proteins were utilized for routine pathologic evaluation: glial fibrillary acidic protein (1:400, Dako M076101), synaptophysin (1:400, Leica MCL-L-SYNAP-299), NEU-N (1:5000, Chemicon MAB377), neurofilament protein (1:100, Dako M076229), microtubule-associated protein 2 (MAP2 1:10,000, Sigma M4403), and Ki67 (1:200, Dako M7240).
8
Multimodal Histopathological Analysis
9
Immunohistochemical Analysis of Brainstem Slices
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Slices used for patch-clamp analysis or fresh brainstem slices (~200 µm thick) were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Free-floating slices were blocked in 4% goat serum and 0.3% (w/v) Triton X-100 in PBS for 1 hr and then were incubated with primary antibody overnight at 4°C. The following primary antibodies were used: mouse anti-Olig1 (1:500; Millipore, MAB5540), mouse anti-MAP2 (1:200; Millipore, MAB3418), mouse anti-NeuN (1:200; Millipore, MAB377), rabbit anti-GFAP (1:500; DAKO, Z033429), mouse anti-CC1 (1:200, Millipore, OP80), mouse anti-NeuN (1:600, Millipore, MAB377), anti-Olig2 (1:100, Abcam, 109186), rat anti-PDGFRa (1:300, abcam, AB90967), rabbit anti-BDNF (1:100; Bioss, BS4989R), mouse anti-TrkB (1:50; Santa Cruz, sc-136990), and guinea pig anti-VGluT1 (1:1000; Millipore, AB5905). Tissues were then incubated with different Alexa-conjugated secondary antibodies (1:500; Invitrogen) for 2 hr at room temperature. After three rinses with PBS, slices were coverslipped using mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vectashield; Vector Laboratories) to counterstain cell nuclei. Stained slices were viewed on a confocal laser-scanning microscope (Zeiss LSM-510) at 488, 568, and 633 nm using 40 × or 60 × oil immersion objective.
10
Characterizing Neuronal and Astroglial Phenotypes
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Stylets (n = 3) were collected in the surgical room and their tip was immediately immersed in fixative solution and cell block preparation was performed using the Shandon™ cytoblock™ preparation system (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. Immuno-histofluorescence staining was performed on de-paraffinized BTIs 10 μm thick sections using antigen retrieval treatment and with secondary antibodies coupled with Alexa fluorochrome (A488 or A555). Neuronal and astroglial expression were identified using β3-tubulin (Covance, PRB435P) or NeuN (Millipore, MAB377) antibodies, and GFAP (DAKO, Z334) antibody, respectively. Further neuronal phenotypic characterization was investigated using antibody against: tyrosine hydroxylase (TH, Chemicon, AB152), vesicular glutamate transporter 1 for glutamatergic neurons (V-Glut1, Chemicon, MAB5502), glutamic acid decarboxylase-67 (GAD-67, Chemicon MAB5406) for GABA-ergic neurons. Sections were then counterstained with DAPI to label cell nuclei. Images were obtained using a fluorescence microscope Zeiss 5.1 (Zeiss, Iena, Germany) coupled to a digital camera and Axiovision 4.8 software (Zeiss).
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