To verify the injection sites, coronal sections (18 μm) were cut on a cryostat (Leica, Germany). Synthesis of riboprobes and in situ hybridization (ISH) was performed as described previously [11] (link). Briefly, sections were fixed in 4% paraformaldehyde (10 m) and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (10 m). After prehybridization (2 h) in hybridization solution (containing 50% deionized formamide, 5XSSC, Denhardt's solution, 250 μg/ml tRNA Baker's yeast and 500 μg/ml sonificated salmon sperm DNA), 150 μl hybridization mixture with 400 ng/ml digoxigenin labeled riboprobe against full-length eGFP mRNA (DQ768212) was applied to each slide and slides were incubated overnight at 68°C. Subsequently, the slides were quickly washed in 2XSSC, followed by a 2 h wash in 0.2XSSC, both at 68°C, followed by a 1 h incubation in 10% fetal calf serum in 0.1 M Tris pH 7.5/0.15 M NaCl. Digoxigenin was detected by an alkaline phosphatase labeled antibody (1∶5000; Roche, Germany) using nitroblue tetrazolium and bromochloroindolylphosphate as a substrate. Finally, sections were dehydrated in ethanol, cleared in xylene and mounted with Entallan (Merck, Germany). Pictures of the injection sites were digitalized using a MCID microscope (Zeiss, Germany).
Entallan
Manufactured by Merck Group
Sourced in Germany, Netherlands
Entallan is a laboratory product manufactured by Merck Group. It is a mounting medium used for the mounting and preservation of microscope slides. Entallan is a transparent, non-fluorescent liquid that dries to form a durable and clear mounting medium.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Alkaline phosphatase labeled antibody, by Roche (1 mentions)
Cryostat, by Leica (1 mentions)
Scn400f whole slide scanner, by Leica (1 mentions)
Picric acid, by Merck Group (1 mentions)
Sirius red, by Polysciences (1 mentions)
Axioskop 2 light microscope, by Zeiss (1 mentions)
Nbt bcip, by Roche (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Dako mounting medium, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit antibody, by Merck Group (1 mentions)
Anti brdu antibody, by Agilent Technologies (1 mentions)
Axioplan, by Zeiss (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Lithium carbonate solution, by Merck Group (1 mentions)
Nanozoomer digital slide scanner, by Hamamatsu Photonics (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Luxol fast blue solution, by Merck Group (1 mentions)
Luxol fast blue (lfb), by Merck Group (1 mentions)
6 protocols using entallan
1
Verification of Injection Sites via ISH
2
Quantification of Myocardial Fibrosis
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To evaluate the extent of fibrosis, sections were fixed with 4% paraformaldehyde in PBS, stained with PicroSirius Red and examined by light microscopy. Briefly, paraffin embedded sections were deparaffinated and rehydrated. After repeated washes, slides were incubated in PicroSirius Red working solution pH 2.0 (Picric acid, Sigma-Aldrich (Zwijndrecht, Netherlands), 74069; Sirius Red, Polysciences, Inc. (Hirschberg an der Bergstrasse, Germany) C.I. 35780) for 60 min. Sections were directly transferred to 0.01 M HCl solution and incubated under constant movement. Sections were dehydrated again and a coverslip was placed using Entallan (Merck, Schiphol-Rijk, Netherlands, 107960). Stained sections were scanned at a 40× magnification on a Leica SCN400F Whole Slide Scanner. Quantification of healthy myocardium was performed by computed analysis of the percentage red stained tissue with Image J (NIH), which refers to collagen. Regions of interest were selected manually by lining the tissue of both ventricles and septum. The subepicardial layer was defined as 0.2 mm tissues underneath the epicardial layer. By means of a macro the fraction of red staining to the total area was calculated.
3
Non-radioactive In Situ Hybridization of Sema7A and β1-integrin
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Non-radioactive in situ hybridization was performed on coronal brain sections (16 μm) of adult mice using digoxigenin-labelled RNA probes targeting Sema7A mRNA (rat Sema7A; NC_005107.4, nt 205–941 of coding region) or β1-integrin mRNA (mouse β1-integrin; NM_010578, nt 603–1205 of codion region). Probe design and synthesis, and non-radioactive in situ hybridization were performed as described previously3 (link). In brief, sections were hybridized with 400 ng ml−1 RNA in 150 μl hybridization mixture containing 50% deionized formamide, 5 × standard saline citrate (SSC), 1 × Denhardt's solution, 0.25 mg ml−1 sonificated salmon sperm DNA, and 0.5 mg ml−1 tRNA baker's yeast. After overnight hybridization at 55 °C, slides were washed, incubated with anti-digoxigenin antibodies (1:2,500, 1093274 Boehringer Mannheim) and developed with BCIP/NBT (Boehringer Mannheim). Subsequently, sections were dehydrated and embedded in Entallan (Merck). For co-labelling of BrdU and Sema7A, 800 ng ml−1 RNA probe was used and Triton X-100 was omitted during antibody incubation steps. Sections were embedded in 95% glycerol to avoid loss of in situ hybridization signal. Representative sections were digitized using an Axioskop2 light-microscope (Zeiss).
4
Immunohistochemical Detection of 5-mC and 5-hmC
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The FFPE tissue sections, pretreated as described above, were incubated with the primary antibodies mouse monoclonal anti 5-mC diluted 1:500 [OptimAnti-5-Methylcytosine (33D3) Eurogentec BI-MECY-0100)] or rabbit monoclonal anti 5-hmC diluted 1:500 [Recombinant Anti-5-hydroxymethylcytosine antibody (RM236) (ab214728)] at 37 °C for 30 min, washed three times for 5 min with PBT and incubated at 37 °C for 30 min with Biotinylated Horse anti-Mouse immunoglobulin (IgG) (Vector Laboratories, BA-2001 Burlington, Canada) or Biotinylated Goat anti-Rabbit IgG, (Vector Laboratories, BA-1000 Burlington, Canada) applied in a 1:200 dilution in PBT with 5% normal goat serum. After washing twice for 5 min with PBT and once for 5 min with PBS, samples were incubated at 37 °C for 30 min with ABC complex, diluted 50× in PBS (Vector Laboratories, PK-6010 Burlingame, CA, USA). Sections were washed and incubated with freshly prepared diaminobenzidine (DAB) (liquid DAB + substrate chromogen system; DAKO, K3467) for 7 min at room temperature and subsequently washed with MilliQ water. The sections were counterstained with hematoxylin (hematoxylin solution modified according to Gill II, Merck, 1.05175.0500), dehydrated in an ascending ethanol series, and mounted with Entallan (Merck 1.07961.0100).
5
Immunohistochemical Analysis of Glioblastoma
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Glioblastoma cells were stained as published before [104 (link)]. Ki67 was used for the assessment of proliferation, and cleaved caspase 3 for apoptosis detection (Table 1 ). The labeling of incorporated BrdU was visualized with an anti-BrdU antibody (1:100, DAKO, Aligent, Santa Clara, CA, USA) (Table 1 ), which was applied for 1 h. For all antibodies and all immunohistochemical stainings, the subsequent steps were identical. After washing with PBS, a biotinylated goat anti-rabbit antibody (1:100, Sigma Aldrich) was applied for one hour, the cells were washed three times with PBS and incubated with Streptavidin for one hour (1:100, Sigma Aldrich). After washing with PBS and Tris buffer, the slides were stained with DAB (Sigma Aldrich) and Hematoxylin (Merck Millipore) and covered with Entallan (Merck Millipore). Propidium iodide-labeled cells were additionally stained with Sytox Green (1:10 000, S7020, Thermo Fisher) for 5 min before covering with DAKO mounting medium (DAKO).
For assessing cell death and proliferation at least 100 cells were counted and 5 areas for each cover slip were recorded with an Axioplan (Zeiss, Oberkochen, Germany) and analyzed using ImageJ v1.46r (National Institutes of Health, Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI, USA).
For assessing cell death and proliferation at least 100 cells were counted and 5 areas for each cover slip were recorded with an Axioplan (Zeiss, Oberkochen, Germany) and analyzed using ImageJ v1.46r (National Institutes of Health, Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI, USA).
6
Luxol Fast Blue Staining Protocol
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Paraffin sections seven mm in thickness were collected on glass slides (Superfrost Plus, VWR international, Leuven, Belgium) and dried at 37 C. Sections were deparaffinized and hydrated to 95% ethanol and incubated in luxol fast blue solution (0.1% w/v luxol fast blue, Sigma-Aldrich, in 95% ethanol) overnight at 55 C. The sections were then quickly rinsed in 95% ethanol and incubated in lithium carbonate solution (0.05% w/v, Sigma-Aldrich, in distilled H 2 O) for 30 s. Next, the samples were rinsed in 70% ethanol and subsequently in ddH2O. The sections were dehydrated to 100% ethanol and mounted with Entallan (Merck). Whole slides were digitalized using a NanoZoomer digital slide scanner (Hamamatsu), and the slides were analyzed using Hamamatsu software NDPview2.
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