Unstained 3-μm-thick slides of formalin-fixed, paraffin-embedded (FFPE) tissues were obtained and submitted for immunostaining using an automated stainer (Dako Omnis, Glostrup, Denmark). The following primary antibodies were used: Glial Fibrillary Acidic Protein (GFAP) (1:200, clone 6F2, Dako, Glostrup, Denmark), Olig2 (1:500, clone OLIG2, Sigma-Aldrich, Saint-Louis, USA), SOX10 (1:50, clone A-2, Diagomics, Blagnac, France), neurofilament (1:100, clone NF70, Dako, Glostrup, Denmark), NeuN (1:1000, clone A60, Sigma-Aldrich, Saint-Louis, USA), synaptophysin (1:150, clone Synap, Dako, Glostrup, Denmark), chromogranin A (1:200, clone LK2 H10, Diagnostic Biosystem, Pleasanton, USA), EMA (1:200, clone GM008, Dako, Glostrup, Denmark), L1CAM (1:500, clone UJ127.11, Sigma-Aldrich, Saint-Louis, USA), NFκB (1:6000, clone D14E12, Cell Signaling Technology, Danvers, USA), H3K27me3 (1:2500, polyclonal, Diagenode, Liege, Belgium), and Ki-67 (1:200, clone MIB-1, Dako, Glostrup, Denmark). External positive and negative controls were used for all antibodies and staining. MIB-1 labeling index was jointly estimated by two neuropathologists in a hot-spot area.
Clone a60
Manufactured by Merck Group
Sourced in United States, Denmark
The Clone A60 is a laboratory instrument designed for the amplification and cloning of DNA sequences. It is a compact and versatile device that can be used in a wide range of molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Clone mib 1, by Agilent Technologies (2 mentions)
Clone 6f2, by Agilent Technologies (2 mentions)
Clone d14e12, by Cell Signaling Technology (2 mentions)
Clone olig2, by Merck Group (2 mentions)
Clone nf70, by Agilent Technologies (2 mentions)
Clone gm008, by Agilent Technologies (2 mentions)
Clone uj127, by Merck Group (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Automated stainer, by Agilent Technologies (1 mentions)
Reticulin silver plating kit, by Merck Group (1 mentions)
Clone 1a4, by Agilent Technologies (1 mentions)
Clone v9, by Agilent Technologies (1 mentions)
Clone 123c3, by Agilent Technologies (1 mentions)
Ab4069, by Abcam (1 mentions)
Mouse anti γ adaptin, by BD (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Alexa 633 coupled phalloidin, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit cy3, by Dianova (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Ab82812, by Abcam (1 mentions)
11 protocols using clone a60
1
Immunohistochemical Analysis of FFPE Tissues
2
Immunohistochemical Profiling of Tissue Samples
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Unstained 3-μm-thick slides of formalin-fixed, paraffin-embedded tissues were obtained and submitted for immunostaining with an automated stainer (Dako Omnis, Glostrup, Denmark). The following primary antibodies were used: CD56 (pre-diluted, clone 123C3, Dako, Glostrup, Denmark), Glial Fibrillary Acidic Protein (GFAP) (1:200, clone 6F2, Dako, Glostrup, Denmark), Olig2 (1:500, clone OLIG2, Sigma-Aldrich, Saint-Louis, USA), vimentin (1:800, clone V9, Dako, Glostrup, Denmark), neurofilament (1:100, clone NF70, Dako, Glostrup, Denmark), NeuN (1:1000, clone A60, Sigma-Aldrich, Saint-Louis, USA), synaptophysin (1:150, clone Synap, Dako, Glostrup, Denmark), EMA (1:200, clone GM008, Dako, Glostrup, Denmark), CK18 (1:200, clone 6F2, Dako, Glostrup, Denmark), smooth muscle actin (1:4000, clone 1A4, Dako, Glostrup, Denmark), NFκB (1:6000, clone D14E12, Cell Signaling Technology, Danvers, USA), L1CAM (1:500, clone UJ127.11, Sigma-Aldrich, Saint-Louis, USA), and Ki-67 (1:200, clone MIB-1, Dako, Glostrup, Denmark). Reticulin staining was performed using the Reticulin silver plating kit according to Gordon & Sweets (Merck Millipore, Guyancourt, France). External positive and negative controls were used for all antibodies and staining.
3
Immunohistochemical analysis of Radixin
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The following primary antibodies were used: rat anti-radixin (R21, gift from S. Tsukita, WB 1:50, ICC 1:50); rabbit anti-radixin (Sigma-Aldrich, #R3653, IHC 1:200); rabbit anti-radixin (Abcam, EP 1862Y, #ab52495, IHC 1:200); mouse anti-γ-adaptin (BD Biosciences, #610386; WB 1:5,000); mouse anti-ezrin (Abcam, #ab4069, clone 3C12, WB 1:1,000); mouse anti-neuN (Millipore, clone A60, #MAB377, IHC 1:1,00). The following secondary antibodies were used: peroxidase-conjugated donkey anti-rabbit (Dianova, Hamburg, Germany, #711-036-152, WB 1:10,000); peroxidase-conjugated donkey anti-rat (Dianova, #712-036-153, WB 1:10,000); peroxidase-conjugated donkey anti-mouse (Dianova, #715-036-151, WB 1:10,000); IRDye 800CW goat anti-rabbit (LI-COR, IgG, #926-32211, WB 1:10,000); IRDye 680RD goat anti-mouse (LI-COR, IgG, #926-68070, WB 1:10,000); Alexa-488 goat anti-mouse (Dianova, #115-545-146, IHC 1:500); Cy3 donkey anti-rabbit (Dianova, #711-166-152, IHC 1:500); Cy3 donkey anti-rat (Dianova, #712-166-150, IHC 1:500); Atto488-labelled FluoTag-X4 anti-rabbit nanobody (NanoTag, IgG, #N2404, IHC 1:200). Alexa-633-coupled phalloidin (Thermo Scientific, #A22284) or Tritc-coupled phalloidin (Tebu-bio, #PHDR1) was used to visualize actin-containing stereocilia. Diamidino-2-phenylindole (DAPI, Sigma, #D9542) was used to stain the nucleus.
4
Immunohistochemical Analysis of Apoptosis and Cellular Markers
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Immunohistochemistry was performed principally by the following methods previously described27 (link). Sections were pre-treated with Proteinase K before TUNEL staining using ApopTag peroxidase in situ apoptosis detection kit (Millipore). The staining was amplified using biotin-conjugated secondary antibodies, VECTASTAIN ABC system (Vector) and TSA Plus system (Perkin Elmer). Monoclonal anti-BrdU (1:100, Clone 3D4, BD Biosciences, 555627), anti-Calbindin (1:1000, Abcam, ab82812), anti-Tuj1 (1:1,000, Abcam, ab78078), anti-Nestin (1:50, rat-401, DSHB), anti-FLAG M2 (1:50, Sigma, F1804) and anti-NeuN (1:500, Clone A60, Millipore, MAB377) antibodies were used. Polyclonal anti-HSF1X50 (link) (1:500), anti-HSF1 antibody (1:200, C-19, SCBT, sc-8061) and anti-Cutl1 (1:100, M-222, SCBT, sc-13024) antibodies were also used. The HSF1 expression pattern in Supplementary Fig. 5 was consistently obtained by using both HSF1X and HSF1 C-19 antibodies.
5
Microglial-Neuronal Interactions Analysis
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The brains of SAL-injected animals were processed to interrogate microglial-neuronal interactions. Microglia were detected with a rabbit polyclonal antibody against Iba1 (#019-19741; Wako Chemicals). Neurons were detected with a mouse monoclonal antibody against NeuN, clone A60 (MAB377; Millipore). The protocol for all antibodies was similar to that described above, and PBS was used throughout to wash the sections. Sections were blocked with 5% normal donkey serum (NDS) with 0.3% Triton-X in PBS at room temperature for 1 h. Primary antibodies were diluted in a solution of 5% NDS and 0.3% Triton-X in PBS to yield dilutions of 1:1,000 primary antibody for both Iba1 and NeuN. Sections were incubated overnight at 4°C. For fluorescent signal detection, the following Alexa Fluor conjugated secondary antibodies were used: Donkey anti-mouse-488, donkey anti-rabbit-568 (1:500, both purchased from Thermo Fisher Scientific). Sections were incubated in the dark for 2 h at room temperature. Labeled sections were mounted onto glass slides and cover slipped with fluorescent mounting medium (Vector laboratories).
6
Immunohistochemical Analysis of Neuroinflammation
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After PET-measurements at day 42, animals were decapitated under deep anesthesia with isoflurane. The brains were rapidly removed, frozen in 2-methylbutane, and stored at -80°C prior to further histological and immunohistochemical processing. Adjacent serial coronal brain sections were cut at 500 μm intervals (slice thickness 10 μm) and stained with hematoxylin and eosin (H&E) according to standard protocols. The mAb against the complement receptor 3/CD11b identified microglia/macrophages (clone OX42, dilution 1:1000, AbD Serotec, Oxford, UK, cat-# MCA275R). Microglia activation was assessed by staining for MHC class II (clone Ox6, dilution 1:400, AbD Serotec, Oxford, UK, cat-#MCA46G). Phagocytic cells were identified with mAb ED1 (clone ED1, dilution 1:1000, AbD Serotec, Oxford, UK, cat-# MCA341). The neuronal marker NeuN (clone A60, dilution 1:1000, Millipore, Billerica, USA, cat-# MAB377) was used to assess neuronal integrity in the peri-infarct zone, in order to exactly distinguish infarcted from intact tissue. For visualization, we used the ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (Sigma, Munich, Germany) or Vector SG substrate kit for peroxidase (Vector laboratories, Burlingame, CA, USA.) for anti-NeuN staining as the final reaction product.
7
Immunohistochemical Characterization of Neuronal and Glial Markers
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Primary antibodies used were rabbit anti-HSV-1 (B0114, Dako), chicken anti-NeuN (ab134014, Abcam), mouse anti-NeuN (clone A60, Millipore), and rabbit anti-beta III Tubulin (ab18207, Abcam), rabbit anti-Iba1 (Wako), rabbit anti-STAT1α91 (M-23, Santa Cruz Biotechnology). The mouse anti-A5 antibody (FE-A5), developed by Bruce A. Fenderson at Thomas Jefferson University, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA. Secondary antibodies used were goat-anti-mouse/rabbit Alexa 555, goat-anti-mouse/rabbit Alexa 488 (Invitrogen), and donkey-anti-chicken Alexa488 (Jackson ImmunoResearch Laboratories). Isolectin B4 conjugated to FITC (Sigma) was added to cultures at a concentration of 10μg/mL to stain KH10 neurons in chambers [67 ]. Counterstaining was done by incubation with DAPI (Invitrogen). Samples were mounted using FluorSave Reagent (Calbiochem). When indicated, cells were treated with 12.5U/mL (unless noted) IFNβ (PBL Interferon Source), 100ng/mL IFNγ (Miltenyi Biotec) or 100ng/mL IFNλ2 (PeproTech) for 18 hours prior to infection or for the specified amount of time prior to staining.
8
Comprehensive Neuronal and Glial Marker Profiling
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All reagents for immunohistochemistry (IH) and western blotting (WB) were of the highest available grade. Antibodies and dilutions included rabbit anti-Wwox (1:1000 for IH and WB, HPA050992, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-neuronal nuclei (NeuN) (1:2000 for IH, clone A60, Millipore, Billerica, MA, USA), rabbit anti-NeuN (1:2000 for WB, clone EPR1263, Abcam plc, Cambridge, UK), rabbit anti-microtubule associated protein 2 (MAP2) (1:1000 for IH and WB, Abcam), FluoroPan Neuronal Marker (1:50 for IH, Millipore), mouse anti-myelin basic protein (MBP) (1:50 for IH and WB, clone 1, Millipore), mouse anti-2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) (1:500 for IH and WB, clone 11-5B, Sigma-Aldrich), mouse anti-adenomatous polyposis coli (APC) (1:1000 for IH, clone CC-1, Millipore), goat anti-glial fibrillary acidic protein (GFAP) (1:2000 for IH, 1:1000 for WB, Abcam), goat anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:2000 for IH, Abcam), rabbit anti-Iba1 (1:1,000 for WB, Wako Pure Chemical Industries, Ltd., Tokyo, Japan), and mouse anti-β-actin (1:5000 for WB, Applied Biological Materials Inc., Richmond, BC, Canada).
9
Immunocytochemistry of Na+ Channels in Neuro2a
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The round microscope coverslips with Neuro2a were removed from 6-well plates to be fixe for 60 min in a paraformaldehyde (PFA) solution and immediately used for immunocytochemistry. The fixed cells were incubated overnight at 4°C in PBS 1X containing 5% native goat serum, 1% BSA with rabbit anti-NaX antibody targeting the interdomain 2–3 region of the NaX channel's α-subunit (1/250) and mouse anti-NeuN (1/500, Clone A60, Millipore) or mouse anti-Na+/K+-ATPase α3 subunit (1/10, Clone XVIF9-G10, Sigma-Aldrich) or mouse anti-Na+/K+-ATPase α1 subunit (1/10, Clone C464.6, Millipore) or chicken anti-MAP-2 antibody (1/100, Polyclonal Antibody, Millipore). The cells were first washed in PBS and incubated for 2 h at room temperature in PBS 1Xcontaining AlexaFluor-488 goat anti-rabbit (1/500, green, Invitrogen), and AlexaFluor-555 goat anti-mouse (1/500, red, Invitrogen) or AlexaFluor-555 goat anti-chicken (1/500, red, Invitrogen) as secondary fluorescent antibodies to visualize the NaX and NeuN or Na+/K+-ATPase α3 or α1 isoforms or MAP-2 proteins, respectively. The specificity of the NaX antibody has been tested using SCN7A control peptide (Cedarlane; 68912-12) at different concentration from 0 to 10 μg in differentiated Neuro2a cells (see Supplemental Figure 1 ).
10
Quantifying Hippocampal and MEC Lesions
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The brains were prepared to track the hippocampal tetrode locations in cresyl violet stained sections and to quantify the MEC lesion extent in NeuN (1:15000, Chemicon, CloneA60) stained sections with the Cavalieri method, as previously described10 . The volume of the spared tissue was estimated for the MEC layer II, MEC layer III, MEC deep layers, dorsal parasubiculum, ventral parasubiculum, and hippocampus. Damage to the brain areas other than MEC was not substantial, as previously reported10 .
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