ICC/IF for Anti-ROBO3 [IPI-ROBO3.124] in binding assay
Addgene #251721
- Data Submitted By
- Alexander Morano , Travis Riedel and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- February 11, 2026
- Publication Date
- March 11, 2026 (modified March 13, 2026)
- DOI
- https://doi.org/10.57733/addgene.k6fbks
- Abstract
- Experimental results for Anti-ROBO3 [IPI-ROBO3.124] (Addgene #251721) in an immunofluorescence (IF) binding assay with ExpiCHO cells expressing human or mouse ROBO3.
- Citation
-
ICC/IF for Anti-ROBO3 [IPI-ROBO3.124] in binding assay. Morano A, Riedel T, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.k6fbks
![A) Representative immunofluorescence (IF) images of ExpiCHO cells transfected with human (green/GFP) and mouse (blue/BFP) ROBO3 and labeled with Anti-ROBO3 [IPI-ROBO3.124] (Addgene #251721) in magenta. Images taken at 40X. Scale bar: 20 µm. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. Anti-ROBO3 [IPI-ROBO3.124] labeling of human (left) and mouse (right) ROBO3 is shown in the top row, and compared to a positive (left) and negative (right) control in the bottom row. For both panels, Anti-ROBO3 [IPI-ROBO3.124] was used at a 1 µg/mL (1:1,000 dilution).](https://datahub-media.addgene.org/report-images/k6fbks/ROBO3.124_IF_Binding_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-ROBO3 [IPI-ROBO3.124]
Addgene #251721 -
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 ROBO3
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-ROBO3 [IPI-ROBO3.124]
- Source
- Addgene
- Catalog Number
- 251721
- Host Species
- Rabbit
- Target Antigen
- ROBO3
- 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- 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 Anti-ROBO3 [IPI-ROBO3.124] 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 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 and mouse ROBO3.