For immunocytochemical staining, cells were washed twice in PBS, fixed in 4% paraformaldehyde for 5 min and then covered with 70% ethanol for storage at −20°C. When immunostained, cells were rehydrated in PBS and then stained with antibodies as described previously [11 (link)]. Primary antibodies were used at a dilution of 1:500, except for Mago (1:250) and eIF4A3 (antibodies a and b at 1:250 and 1:2000 respectively). Alexa fluorophore-conjugated secondary antibodies (Thermo-Scientific, Life Technology) were used at a dilution of 1:500. Fluorescently labelled cells were viewed using Zeiss Axioscope fluorescence microscope (Zeiss, Cambridge, U.K.), and images were captured using an Axiocam digital camera system (Zeiss) and Axiovision image analysis software (version 4.7, Zeiss). Images from immunocytochemistry were analysed on ImageJ and ratio of cytosolic over nuclear fluorescence was obtained by subtracting the background fluorescence measured in several points next to each cell. The corrected total cell fluorescence (CTCF) intensity is measured according to the formula: CTCF = integrated intensity – (area of selected cell × mean fluorescence of background readings).
Axiocam digital camera system
Manufactured by Zeiss
Sourced in Germany
The AxioCam digital camera system is a high-performance imaging solution designed for microscopy applications. It captures detailed images with exceptional clarity and resolution. The AxioCam utilizes advanced digital imaging technology to provide reliable and accurate data acquisition for a wide range of microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision le, by Zeiss (3 mentions)
Axio vert a1 inverted microscope, by Zeiss (3 mentions)
Axiophot microscope, by Zeiss (2 mentions)
Axiovision 3, by Zeiss (2 mentions)
Axioskop microscope, by Zeiss (2 mentions)
Alexa fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axiovision image analysis software, by Zeiss (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Axioscope microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Axioimager a1m microscope, by Zeiss (1 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (1 mentions)
10 protocols using axiocam digital camera system
1
Immunocytochemical Staining Protocol
2
MTT Assay for Cell Viability
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To exclude possible effects of α-HPy and α-CJe on cell viability, a so called MTT-assay was performed (Levitz and Diamond 1985 (link)). For this, to SiMa cells pretreated with either of both antisera or to untreated controls, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Merck, Darmstadt, Germany) was added at a final concentration of 50 μg/ml, followed by a 2-h incubation at 37 °C under a humidified atmosphere with 5% CO2. After this, brightfield photographs of the stained cells were taken with an Axiocam digital camera system, mounted on an Axiophot microscope (Zeiss, Jena, Germany). Photographs were then evaluated densitometrically with the FiJi clone of the open source image analysis program ImageJ (see https://imagej.net ), followed by statistical analysis of the obtained results with the free OpenOffice Calc software (see https://www.openoffice.de ).
3
Immunocytochemistry Protocol for Cell Analysis
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Immunocytochemistry was performed as described earlier (Dahm et al. 2010 (link)). In brief, cells on glass cover slips (Menzel, Braunschweig, Germany) were washed with PBS and fixed for 10′ with 4% paraformaldehyde (PFA) in PBS. After washing, cells were permeabilized for 10′ with a mixture of acetone/methanol (1:1) at − 20 °C. Following three washes with phosphate buffered saline (PBS), cells were blocked for 1 h with goat serum (GS) diluted 1:50 in PBS (PBS-GS). Primary antibodies diluted 1:50 in PBS-GS were applied overnight at 4 °C, followed by three washes with PBS and a 90′ incubation with Atto488-coupled secondary antibodies (Sigma-Aldrich, Steinheim, Germany), diluted 1:400 in PBS-GS at 37 °C. After three washes with PBS, cells were mounted on standard microscope slides using a commercial mounting medium (DAKO, Glostrup, Denmark). Imaging was performed using an Axiocam digital camera system, mounted on an Axiophot microscope (both Zeiss, Jena, Germany).
4
Immunohistochemical Evaluation of Wnt Signaling
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IHC was performed using a combination of pressure cooker heating and the ZytoChem-Plus HRP Polymer-Kit with DAB as chromogenic substrate according to a previous publication by our lab (Scholz et al. 2012 (link)). The primary antibodies in our immunohistochemical staining were anti-β-catenin IgG, anti-E-cadherin IgG (Merck, Darmstadt, Germany) and anti-snail/ slug IgG (Abcam, Cambridge, UK) (Table 1 ). Evaluation, imaging and storing was done with an AxioScope microscope (Carl Zeiss), an AxioCam digital camera system (Carl Zeiss) and the AxioVision software (Carl Zeiss). Immunohistochemical staining was assessed semiquantitatively, according to Remmele and Steger (Remmele and Stegner 1987 (link)) using the IHC score (mean ± SEM). Expression of Wnt signaling markers was captured in different subcellular locations (β-catenin: membrane and plasma, snail/ slug: plasma, E-cadherin: membrane and plasma).
5
Immunofluorescence Staining of Adherent Cells
6
Immunocytochemical Identification of CTCs
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After the CellCollector application was finished, the CellCollector was gently washed in washing buffer, followed by incubation in PBS containing 2% (w/v) bovine serum albumin (BSA) for 30 min at room temperature. CTCs captured by the CellCollector were identified by immunocytochemical staining for EpCAM or cytokeratins 8, 18, and 19. Cells attached to the wire were incubated with a FITC-conjugated mouse monoclonal antibody directed to EpCAM (Acris, clone HEA125-FITC) and an APC-conjugated rabbit antibody raised against CD45 (Exbio, clone MEM-28-Alexa647). Cells were counterstained with the nuclear dye Hoechst33342 (Sigma). Intensity of the immunocytochemical staining of CTC was evaluated using an Axio Imager.A1m microscope (Zeiss, Jena, Germany) equipped with an AxioCam digital camera system and the AxioVision 4.6 software (Zeiss). EpCAM/cytokeratin-positive cells should had additional features, including a large cell body (diameter 10–50 μm), an irregular cell shape, a large irregularly shaped nucleus, and a high nuclear to cytoplasmic ratio. Cells were enumerated on each CellCollector by an operator who was blinded to the clinical information of the patients.
7
FluoroJade-B Staining of Neurodegeneration
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Whole brains were removed and allowed to post-fix in 4% PFA overnight at 4 ℃ before being rinsed with PBS and transferred to a 30% sucrose solution for overnight cryoprotection. Thirty micrometer thick coronal sections were sliced on a freezing stage microtome and stored at 4 ℃ in PBS for staining. FluoroJade-B (FJB) staining was performed using 3 hippocampal slices per animal and batch processed. Briefly, sections were mounted on glass slides, dried, and immersed in a descending ethanol series (100% for 3 min, and 90%, 70%, and dIH2O for 1 min each). Slices were blocked in 0.06% KMnO4 for 30 min, followed by a 2 min rinse in dIH2O, and then incubated in 0.001% FJB solution for 15 min. Slides were rinsed 5 times with dIH2O for 1 min each, dehydrated in an ascending ethanol series (70%, 90%, and 100% for 3 min each), and coverslipped with DPX mounting media. Digital images were obtained with a Zeiss Axioskop microscope equipped with an AxioCam digital camera system and AxioVision 3.0 software (Zeiss, Oberkochen, Germany). Three hippocampal subregions (CA1, CA3, and Hilus, both ipsilateral and contralateral to the KA injection) were scored for positive or negative FJB staining by two independent reviewers who were blinded to treatment group.
8
FluoroJade-B Staining of Neurodegeneration
9
Immunofluorescence Staining of Adherent Cells
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Adherent cells were rinsed with cold 1× PBS and fixed with 2% paraformaldehyde solution for 10-15 min at room temperature (RT), permeabilized with ice-cold 100% ethanol for 5 min, rinsed again with 1× PBS, and blocked overnight at 4°C with 5% normal goat serum (NGS; Thermo Fisher). Cells were incubated for 2 h at RT with primary antibodies, then rinsed three times with 1× PBS to remove excess antibody. Cells were incubated with fluorescently conjugated secondary antibodies for 1 h at RT in the dark. Cells were washed three more times with 1× PBS. Nuclear counterstaining was performed using Vectashield mounting media with DAPI (Vector Laboratories). For more information on the antibodies used, please refer to Tables S1 and S2 . Cells were imaged with a phase contrast and fluorescence AxioVert A1 inverted microscope (Zeiss) equipped with AxioCam digital camera system (Zeiss). Images were exported using AxioVision LE (Zeiss) and merging was performed in Adobe Photoshop.
10
Immunofluorescence Staining of Adherent Cells
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Adherent cells were rinsed with cold PBS and fixed with 2% para-formaldehyde solution for 15 min at room temperature, permeabilized with ice-cold 100% methanol for 2 min, rinsed again with 1x PBS, and blocked for 1 hr with 5% normal goat serum (NGS; Thermo Fisher). Cells were incubated overnight with primary antibodies, then rinsed three times with 1 × PBS to remove excess antibody. Cells were incubated with fluorescently conjugated secondary antibodies for 1 h at RT in the dark. Cells were then washed three more times with 1 × PBS. Nuclear counterstaining was performed using Hoecht. Cells were imaged with a phase contrast and fluorescence AxioVert A1 inverted microscope (Zeiss) equipped with AxioCam digital camera system (Zeiss). Images were exported using AxioVision LE (Zeiss) and merging was performed in Adobe Photoshop.
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