ICC/IF for Anti-NTN1 [IPI-NTN1.45 ] in binding assay
Addgene #259427
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- July 27, 2026
- Publication Date
- August 05, 2026 (modified August 14, 2026)
- DOI
- https://doi.org/10.57733/addgene.6ikbwd
- Abstract
- Experimental results for Anti-NTN1 [IPI-NTN1.45 ] (Addgene #259427) in an immunofluorescence (IF) binding assay with ExpiCHO cells expressing human or mouse NTN1.
- Citation
-
ICC/IF for Anti-NTN1 [IPI-NTN1.45 ] in binding assay. Morano A, Riedel T, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.6ikbwd
![Immunofluorescence (IF) of ExpiCHO cells transfected with human NTN1 and labeled with Anti-NTN1 [IPI-NTN1.45] (Addgene #259427). Left image shows three channels: IPI-NTN1.45 (magenta), GFP transfection control (green), and DAPI (blue). Right image only shows IPI-NTN1.45 and DAPI. Scale bar = 20 µm. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. Anti-NTN1 [IPI-NTN1.45] labeling of human (left) and mouse (right) Anti-NTN1 [IPI-NTN1.45] is shown in the top row, and compared to a positive (left) and negative (right) control in the bottom row. For both panels, Anti-NTN1 [IPI-NTN1.45] was used at a 1 µg/mL (1:1,000 dilution).](https://datahub-media.addgene.org/report-images/6ikbwd/NTN1.45_IFBinding_v2.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-NTN1 [IPI-NTN1.45]
Addgene #259427 -
Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
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 V5-tagged human NTN1
Buffer
- Name
- PBS
Fixative
- Fixative
- Formaldehyde/Paraformaldehyde
- Time
- 15 min
- Temperature
- Room Temperature
Permeabilization
- Agent
- Triton X-100 in PBS
- Concentration
- 0.20%
- 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-NTN1 [IPI-NTN1.45]
- Source
- Addgene
- Catalog Number
- 259427
- Host Species
- Rabbit
- Target Antigen
- NTN1
- Concentration
- 1 µg/mL (1:1,000 dilution)
- Time
- 60 min
- Temperature
- Room Temperature
Secondary Antibody
- Name
- Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
- 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 Anti-NTN1 [IPI-NTN1.45 ] labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 22000 (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 Isotype Control [IPI-MOPC-21] (Addgene #237822) used to confirm lack of nonspecific binding to the sample.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- The antibody shows binding to human NTN1.