Immunostaining was performed following deparaffinization and rehydration of slides. Antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Nonspecific binding was blocked using Vector mouse IgG blocking serum 30 min at room temperature. Samples were incubated at room temperature with rabbit monoclonal antibodies cleaved caspase 3, and Ki67. Slides were developed with Vector Immpress rabbit IgG and Vector Immpress mouse IgG (Vector Laboratories) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (Vector Laboratories) for 3 min. Slides were counterstained with hematoxylin. A 3DHistech MIDI Scanner (Perkin Elmer) was used to capture whole slide digital images with a 20x objective. Images were converted to into MRXS files and computer graphic analysis was completed using inForm 1.4.0 Advanced Image Analysis Software (Perkin Elmer).
Inform 1.4.0 advanced image analysis software
Manufactured by PerkinElmer
InForm 1.4.0 Advanced Image Analysis Software is a powerful tool for analyzing digital images. It provides a comprehensive set of image analysis capabilities, enabling users to extract and quantify relevant information from their images. The software is designed to handle a wide range of image types, including microscopy, histology, and immunohistochemistry samples.
Automatically generated - may contain errors
Lab products found in correlation
Vector immpress rabbit igg, by Vector Laboratories (3 mentions)
Vector immpress mouse igg, by Vector Laboratories (3 mentions)
Vector immpact dab, by Vector Laboratories (3 mentions)
Mitotracker orange, by Thermo Fisher Scientific (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
4 protocols using inform 1.4.0 advanced image analysis software
1
Immunostaining and Digital Pathology Analysis
2
Immunohistochemical Evaluation of Cell Signaling
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Immunostaining was performed following deparaffinization and rehydration of slides. Antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Nonspecific binding was blocked using Vector mouse IgG blocking serum for 30 min at room temperature. Samples were incubated at room temperature with rabbit monoclonal antibodies pS6 (Cell Signaling Technologies, 5364), cleaved caspase 3 (Cell Signaling Technologies, 9661), and Ki67 (Dako, M7240). Slides were developed with Vector Immpress rabbit IgG (Vector Laboratories, MP7401) and Vector Immpress mouse IgG (Vector Laboratories, MP7400) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (Vector Laboratories, SK4105) for 3 min. Slides were counterstained with hematoxylin. A 3DHistech MIDI scanner (Perkin Elmer) was used to capture whole-slide digital images with a 20× objective. Images were converted to MRXS files, and computer graphic analysis was completed using inForm 1.4.0 advanced image analysis software (Perkin Elmer).
3
Immunohistochemical Analysis of Cellular Markers
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Immunostaining was performed following deparaffinization and rehydration of the slides, antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature and nonspecific binding was blocked using 10% normal horse serum for 30 min at room temperature. Samples were incubated at room temperature with p-Y416Src rabbit monoclonal antibody (Y416), cleaved caspase 3, p-Y705Stat3 and Ki67. Slides were developed with Vector Immpress rabbit IgG (#MP7401) and Vector Immpress mouse IgG (#MP7400) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (#SK4105) for 3 min. Sides were counterstained with hematoxylin. A 3DHistech MIDI Scanner (Perkin Elmer) was used to capture whole slide digital images with a 20x objective. Images were converted to into MRXS files and computer graphic analysis was completed using inForm 1.4.0 Advanced Image Analysis Software (Perkin Elmer).
4
Quantifying Mitochondrial Levels in Drug-Treated Cells
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Cells were grown on coverslips and then treated with drug for 24 h. Cells were fixed in 4% paraformaldehyde for 15 min and rinsed with PBS. Cells were washed with a 1% saponin solution for 15 min at room temperature and then washed several times in PBS to remove detergent. Cells were then incubated in Bodipy (ThermoFisher, D3922) at a final concentration of 1 µM for 10 min. Bodipy was removed, and slides were rinsed with PBS, air-dried, and mounted on slides using Dako mounting medium with DAPI.
To detect mitochondrial levels in treated cells, cells were grown on coverslips for 24 h. Mitotracker orange (ThermoFisher, M7511) was diluted in medium with drug at a final concentration of 1 M and incubated overnight. The medium was removed, and cells were fixed with 4% paraformaldehyde for 15 min. Cells were rinsed twice for 5 min in PBS and incubated in cold acetone for 10 min at −20°C. Acetone was removed, and cells were washed in PBS, air-dried, and mounted on slides with Dako mounting medium with DAPI.
A 3DHistech MIDI scanner (Perkin Elmer) was used to capture whole-slide digital images with a 20× objective. Images were converted to MRXS files, and computer graphic analysis was completed using inForm 1.4.0 advanced image analysis software (Perkin Elmer).
To detect mitochondrial levels in treated cells, cells were grown on coverslips for 24 h. Mitotracker orange (ThermoFisher, M7511) was diluted in medium with drug at a final concentration of 1 M and incubated overnight. The medium was removed, and cells were fixed with 4% paraformaldehyde for 15 min. Cells were rinsed twice for 5 min in PBS and incubated in cold acetone for 10 min at −20°C. Acetone was removed, and cells were washed in PBS, air-dried, and mounted on slides with Dako mounting medium with DAPI.
A 3DHistech MIDI scanner (Perkin Elmer) was used to capture whole-slide digital images with a 20× objective. Images were converted to MRXS files, and computer graphic analysis was completed using inForm 1.4.0 advanced image analysis software (Perkin Elmer).
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