ICC/IF for Anti-Netrin-1 [IPI-NTN1.65] in specificity assay
Addgene #241895
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- November 25, 2025
- Publication Date
- November 26, 2025 (modified December 17, 2025)
- DOI
- https://doi.org/10.57733/addgene.4mhwvq
- Abstract
- Experimental results for Anti-Netrin-1 [IPI-NTN1.65] (Addgene #241895) in an immunofluorescence (IF) specificity assay with ExpiCHO cells expressing human or mouse NTNs.
- Citation
-
ICC/IF for Anti-Netrin-1 [IPI-NTN1.65] in specificity assay. Morano A, Riedel T, Moshinsky D 2025. Addgene Report, https://doi.org/10.57733/addgene.4mhwvq
![Each graph depicts the combined quantification of multiple images of the same transfected cells taken at 10X magnification. GFP- or BFP-positive cells were identified and the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing Anti-Netrin-1 [IPI-NTN1.65] (Addgene #241895) labeling, was recorded. IPI-NTN1.65 labeling of human and mouse variants of each netrin family member is compared on the top and bottom rows. To test family-wide cross-reactivity, IPI-NTN1.65 was used at 0.1 µg/mL (1:10000 dilution).](https://datahub-media.addgene.org/report-images/4mhwvq/NTN1.65_IF-SPECIFICITY.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 fragment 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 separately transfected with human NTN1, or human or mouse NTN3, or NTN4. Human NTNs were cotransfected with GFP and mouse NTNs were cotransfected with BFP (transfection controls).
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 fragment 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
GFP- or BFP- 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 16000 (saturation).
Controls
Positive control (not shown): Anti-V5 [IPI-SV5-Pk1] (Addgene #218107) used to confirm IF labeling efficacy (since construct expressing target also has a V5 tag).
Negative control (not shown): 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 only to human Netrin 1.