We performed neuropathologic examinations on biopsied specimens taken from the white matter of anterior horn of lateral ventricle of Patient 5 and 7. Paraffin-embedded fixed tissue sections (7 μm) were stained with hematoxylin & eosin (H&E), and with periodic Schiff. Sections were also immunostained with a DAKO Autostainer system (DAKO, Carpinteria, CA) using EnVision FLEX DAB+ Chromogen with the following antibodies: anti-phosphorylated neurofilament (mouse monoclonal [2836], 1:100; Cell Signaling Technology); anti-CD68 (PGM1) (mouse, 1:100; Dako); anti-CD3 (D7A6E) (rabbit [85061], 1:200; Cell Signaling Technology); anti-CD20 (EP459Y) (rabbit [ab78237], 1:200; abcam); anti-Olig2 (EPR2673) (rabbit [ab109186], 1:100; abcam).
Epr2673
Manufactured by Abcam
EPR2673 is a recombinant monoclonal antibody that recognizes human Mitofusin 2. Mitofusin 2 is an outer mitochondrial membrane protein involved in mitochondrial fusion.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 fluorescent microscope, by Olympus (2 mentions)
Ab15580, by Abcam (2 mentions)
Pe or fitc conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Ab25877, by Abcam (2 mentions)
Af3626, by R&D Systems (2 mentions)
Dako autostainer system, by Agilent Technologies (1 mentions)
Ab109186, by Abcam (1 mentions)
Anti cd68 pg m1, by Agilent Technologies (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoromyelin red, by Thermo Fisher Scientific (1 mentions)
Ab16794, by Abcam (1 mentions)
Anti neurofilament 200, by Merck Group (1 mentions)
Epr12763, by Abcam (1 mentions)
Cy sc002, by Spirochrome (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Magnify chromatin immunoprecipitation system, by Thermo Fisher Scientific (1 mentions)
Nextseq, by Illumina (1 mentions)
Bioruptor ucd 200, by Diagenode (1 mentions)
6 protocols using epr2673
1
Neuropathological Examination of Anterior Horn
2
Immunohistochemical Analysis of Neural Tissue
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Fixed tissue blocks were immersed in 30% sucrose in PBS at 4 °C for 24 h. Later, tissue blocks were frozen using tissue freezing medium (ref. 14,020,108,926, Leica) and finally 30–40 μm slices were obtained in a CM 1860 cryostat (Leica).
Sections were marked with anti-APC antibody [CC-1] (ab16794, abcam) and polyclonal anti- Alpha/Beta-tubulin (ATN02, Cytoskeleton), Anti-Neurofilament 200 (N4142, Sigma), anti-NeuN (EPR12763, Abcam) or anti-Olig2 antibody (EPR2673, Abcam), preceded by heat-induced epitope retrieval in sodium cytrate buffer at 95 °C for 10 min, for nuclear epitopes. After washing with PBS, the sections were stained with the Alexa Fluor 555 A21422, Alexa Fluor 488 A11070 and Alexa Fluor 488 A11015 secondary antibodies (Thermo Fisher Scientific). Nuclear staining was performed with DAPI (62,248, Thermo Fisher Scientific). Myelin sheaths were stained with Fluoromyelin red (F34652, Thermo Fisher Scientific) as indicated in the datasheet. Acute tissue sections were incubated at 37 °C in SiR-tubulin (1:100 mL in HBSS, Spirochrome AG CY-SC002) for one hour prior to imaging.
Sections were marked with anti-APC antibody [CC-1] (ab16794, abcam) and polyclonal anti- Alpha/Beta-tubulin (ATN02, Cytoskeleton), Anti-Neurofilament 200 (N4142, Sigma), anti-NeuN (EPR12763, Abcam) or anti-Olig2 antibody (EPR2673, Abcam), preceded by heat-induced epitope retrieval in sodium cytrate buffer at 95 °C for 10 min, for nuclear epitopes. After washing with PBS, the sections were stained with the Alexa Fluor 555 A21422, Alexa Fluor 488 A11070 and Alexa Fluor 488 A11015 secondary antibodies (Thermo Fisher Scientific). Nuclear staining was performed with DAPI (62,248, Thermo Fisher Scientific). Myelin sheaths were stained with Fluoromyelin red (F34652, Thermo Fisher Scientific) as indicated in the datasheet. Acute tissue sections were incubated at 37 °C in SiR-tubulin (1:100 mL in HBSS, Spirochrome AG CY-SC002) for one hour prior to imaging.
3
Olig2-Bound Chromatin Immunoprecipitation
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Embryonic dorsal forebrains (E14.5) were triturated in Hank’s Balanced Salt Solution (Thermo Fisher Scientific) and fixed in 1% fresh paraformaldehyde in PBS for 5 min at room temperature. Fixation was quenched with 125 mM Glycine at room temperature for 5 min and washed with cold PBS twice. Chromatin immunoprecipitation was performed using MAGnify Chromatin immunoprecipitation system (Thermo Fisher Scientific) with minor modifications as follows. Chromatin was fragmented into 100–300 bp by setting the Bioruptor UCD-200 (Diagenode) to high power and sonicated for 3 rounds of 10 cycles (30 sec ON/30 sec OFF). For immunoprecipitation of Olig2-bound chromatin, 2 μg of anti-Olig2 antibody [EPR2673] (Abcam, ab109186) was incubated with cleared chromatin lysate and 2 μg of whole rabbit IgG was used as a control. Input and ChIPed DNA libraries were prepared using an Illumina Next Seq (single-end reads of 75 bp).
4
Immunohistochemical Analysis of Mouse Brain
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Mouse pups were anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. The brain tissues were embedded in paraffin and sliced coronally in 5 μm. Tissue sections were dewaxed, quenched with 3% hydrogen peroxide for 10 min, and incubated with 5% bovine serum albumin (BSA) for 30 min. The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience). TUNEL staining was performed with the in situ cell death detection kit (Roche). The slides were counterstained with nuclear dye DAPI and observed on an Olympus BX51 fluorescent microscope.
5
Glioblastoma Immunohistochemistry Protocol
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The paraffin‐embedded glioblastoma tissue microarray (TMA) was used for IHC staining,23 and 40 cases of WHO grade IV glioblastomas were further analysed. The slide was performed with de‐paraffinized, rehydrated, and antigen retrieval, then blocked for at least 1 h at 37°C with 1% BSA, and then incubated overnight at 4°C with primary antibodies (anti‐c‐Jun (60A8) Rabbit mAb (1:100, CST, 9165S), anti‐c‐Jun (phospho S63) antibody [Y172] (1:100, Abcam, ab32385), anti‐CD44 Polyclonal antibody (1:100, Proteintech, 15,675‐1‐AP) and anti‐OLIG2 antibody [EPR2673] (1:100, Abcam, ab109186)). After careful washing with 1 × PBS, the slides were incubated with horseradish peroxidase (HRP)‐conjugated antibodies, DAB was used for chromogenic reaction, and the nuclei were counterstained with haematoxylin. The TMA images were taken by Vectra Polaris Automated Quantitative Pathology Imaging System. In addition, we quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. Tissues too small and/or crushed on the TMA were eliminated from analysis.
6
Immunohistochemical Analysis of Mouse Brain
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Mouse pups were anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. The brain tissues were embedded in paraffin, sliced coronally in 5-μm. Tissue sections were dewaxed, quenched with 3% hydrogen peroxide for 10 min, and incubated with 5 % bovine serum albumin (BSA) for 30 min. The sections were stained overnight at 4°C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE-or FITC-conjugated secondary antibodies (eBioscience). TUNEL staining was performed with the in situ cell death detection kit (Roche). The slides were counterstained with nuclear dye DAPI and observed on an Olympus BX51 fluorescent microscope.
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