ICC/IF for Anti-Netrin-1 [IPI-NTN1.65] in binding assay
Addgene #241895
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- November 24, 2025
- Publication Date
- November 26, 2025 (modified December 17, 2025)
- DOI
- https://doi.org/10.57733/addgene.ziscn9
- Abstract
- Experimental results for Anti-Netrin-1 [IPI-NTN1.65] (Addgene #241895) in an immunofluorescence (IF) binding assay with ExpiCHO cells expressing V5-tagged human NTN1.
- Citation
-
ICC/IF for Anti-Netrin-1 [IPI-NTN1.65] in binding assay. Morano A, Riedel T, Moshinsky D 2025. Addgene Report, https://doi.org/10.57733/addgene.ziscn9
![A) Immunofluorescence (IF) of ExpiCHO cells transfected with human NTN1 and labeled with Anti-Netrin-1 [IPI-NTN1.65] (Addgene #241895). Left images show all three channels (IPI-NTN1.65, GFP (transfection control) and DAPI) while the right images only show IPI-NTN1.65 and DAPI. Images taken at 40X magnification. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. IPI-NTN1.65 labeling of human Netrin-1 is shown in the top row, and compared to a positive (left) and negative (right) control in the bottom row. IPI-NTN1.65 was used at 0.1 μg/mL (1:10000 dilution).](https://datahub-media.addgene.org/report-images/ziscn9/NTN1.65_IF-BINDING.png)
Image attribution: Institute for Protein Innovation (IPI)
This report is made available under the Creative Commons Attribution 4.0 International License.
- Antibodies Used
-
Anti-Netrin-1 [IPI-NTN1.65]
Addgene #241895 -
Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG, Fc fragment specific
Jackson ImmunoResearch #111-606-046
Materials & Methods
Sample
- Target Species
- Human
- Cell / Tissue Type
- ExpiCHO
- Additional Information
- ExpiCHO cells were transiently transfected with GFP (transfection control) and human NTN1
Buffer
- Name
- PBS
Fixative
- Fixative
- Formaldehyde/Paraformaldehyde
- Time
- 15 min
- Temperature
- Room Temperature
Permeabilization
- Agent
- Triton X-100 in PBS
- Concentration
- 0.2%
- Time
- 10 min
- Temperature
- Room Temperature
Blocking
- Agent
- Normal goat serum + BSA
- Concentration
- 5% serum, 5% BSA
- Time
- 30 min
- Temperature
- Room Temperature
Primary Antibody
- Name
- Anti-Netrin-1 [IPI-NTN1.65]
- Source
- Addgene
- Catalog Number
- 241895
- Host Species
- Rabbit
- Target Antigen
- NTN1
- Concentration
- 0.1 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Secondary Antibody
- Name
- Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG, Fc fragment specific
- Source
- Jackson ImmunoResearch
- Catalog Number
- 111-606-046
- Concentration
- 2 µg/mL
- Time
- 60 min
- Temperature
- Room Temperature
Additional Information
- Additional Information
Confocal images taken at 40X magnification on an ImageXpress confocal HT.ai microscope.
For quantitative data, GFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing IPI-NTN1.65 labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 12000 (saturation).
Controls
Positive control: Anti-V5 [IPI-SV5-Pk1] (Addgene #218107) used to confirm IF labeling efficacy (since construct expressing target also has a V5 tag).
Negative control: Rabbit IgG used to confirm transfected construct does not interact /produce signal with every antibody.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- The antibody shows binding to human Netrin 1.