To immunostain brain slices, adult mouse brains were infused with Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen and sectioned at 10 µm using a cryostat (HM500-OM, Carl Zeiss, Oberkochen, Germany). The sections were mounted on silane-coated coverslips and dried for ~30 min at room temperature. The samples were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl, pH 7.4) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 10% FCS in PBS, the samples were incubated with the anti-RNG105 antibody over night at 4°C. After washing with PBS, the samples were incubated with Alexa488-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries) for 1 hr at room temperature to label RNG105 and nuclei, respectively. To immunostain cultured neurons, neurons at 12 DIV were fixed and stained in the same way. Fluorescence images were acquired using an A1 confocal laser microscope equipped with a Ti-E inverted microscope (Nikon, Tokyo, Japan) with a 10 × objective lens or a PlanApo VC60 × oil objective lens.
Hm 500 om
Manufactured by Zeiss
Sourced in Germany
The HM 500 OM is a high-quality optical microscope designed for laboratory applications. It features advanced optics and versatile functionality to support a wide range of research and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Axiolab, by Zeiss (2 mentions)
Alexa488 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Plan apo vc 60, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Fujifilm (1 mentions)
Ti e inverted microscope, by Nikon (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
A1 confocal laser microscope, by Nikon (1 mentions)
Tissue freezing medium, by Leica (1 mentions)
Sucrose, by Merck Group (1 mentions)
Image pro plus 4, by Media Cybernetics (1 mentions)
Pannoramic midi, by 3DHISTECH (1 mentions)
4 protocols using hm 500 om
1
Immunostaining of Mouse Brain Slices
2
In Situ Detection of Apoptotic Tumor Cells
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In situ detection of apoptotic cells in the tumors isolated from the mice was performed with a TUNEL assay. The tumors were paraffin embedded and cut into 10-μm-thick sections in a microtome cryostat (HM500 OM, Carl Zeiss, Germany). The TUNEL assay was conducted according to the manufacturer’s protocols. 3,3-Di- aminobenzidine (DAB) was used as the substrate for the peroxidase. Images were captured with a light microscope (Axiolab, Carl Zeiss, Germany), and five images/sample were prepared. Image-Pro Plus 4.5 Software was used to analyze the staining data.
3
Histological Staining of Tissue Samples
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For histological staining, the samples were fixed for 30 min with 4% neutral buffered formalin (Formafix AG) at room temperature and stored, washed with PBS, and processed with sucrose (150 mg/ml and 300 mg/ml; Sigma-Aldrich) before being embedded in tissue freezing medium (Leica). The samples were cut using a cryostat microtome (HM 500 OM; Zeiss) to 8-μm slices and kept at −20 °C until they were being processed for histological staining with safranin O-fast green.
4
Immunohistochemical Analysis of Tumor Samples
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The tumors isolated from the mice were paraffin‐embedded and cut into 10‐μm sections using a microtome cryostat (HM 500 OM; Carl Zeiss, Jena, Germany). Immunohistochemical staining was conducted according to the manufacturer's protocols for the HistostainTM‐Plus Kits. Primary antibodies (as described in the section 2.2. ) were incubated at 4 °C overnight. The images were captured with a light microscope (Axiolab; Carl Zeiss), and five images/sample were prepared. The image‐pro plus 4.5 (Media Cybernetics, Silver Spring, MD, USA) software was used to analyze the staining data.
For the tumor samples from HCC patients, a tissue microarray was created from the 168 HCC samples. After histopathological examination, every sample was excised in one core with a 1.0 mm diameter on one tissue array. Immunohistochemical staining was conducted according to the manufacturer's protocols for the HistostainTM‐Plus Kits. The results were scanned by Pannoramic MIDI (3DHISTECH Ltd., Budapest, Hungary), quantified, and analyzed by Pannoramic viewer andquant center software. For each epitope, the staining score for tumor cells was recorded separately. An H‐score, which reflects the expression level of certain protein, was calculated based on the staining intensity as previously described (Azim et al., 2015 ).
For the tumor samples from HCC patients, a tissue microarray was created from the 168 HCC samples. After histopathological examination, every sample was excised in one core with a 1.0 mm diameter on one tissue array. Immunohistochemical staining was conducted according to the manufacturer's protocols for the HistostainTM‐Plus Kits. The results were scanned by Pannoramic MIDI (3DHISTECH Ltd., Budapest, Hungary), quantified, and analyzed by Pannoramic viewer and
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