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ICC/IF for Anti-Neuroligin-3 [IPI-mNLGN3.3] in binding assay

Addgene #219625

Data Submitted By: Alexander Morano , Travis Riedel and Deborah Moshinsky
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: February 03, 2026
Publication Date: February 09, 2026 (modified February 19, 2026)
DOI: https://doi.org/10.57733/addgene.eeh9jx
Abstract: Experimental results for Anti-Neuroligin-3 [IPI-mNLGN3.3] (Addgene #219625) in an immunofluorescence (IF) binding assay with ExpiCHO cells expressing human or mouse NLGN3.
Citation: ICC/IF for Anti-Neuroligin-3 [IPI-mNLGN3.3] in binding assay. Morano A, Riedel T, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.eeh9jx

**Caption:** A) Immunofluorescence (IF) of ExpiCHO cells transfected with human (top) and mouse (bottom) NLGN3 and labeled with Anti-Neuroligin-3 [IPI-mNLGN3.3] (Addgene #219625). Left images show all three channels (IPI-mNLGN3.3, GFP (transfection control) and DAPI) while the right images only show IPI-mNLGN3.3 and DAPI. Images taken at 40X magnification. B) Combined quantification of multiple images of the same transfected cells taken at 10X magnification. IPI-mNLGN3.3 labeling of human (left) and mouse (right) Anti-Neuroligin-3 [IPI-mNLGN3.3] is shown in the top row, and compared to a positive (left) and negative (right) control in the bottom row. For both panels, IPI-mNLGN3.3 was used at a 1 µg/mL (1:1,000 dilution).
**Image attribution:** Institute for Protein Innovation (IPI)

This report is made available under the Creative Commons Attribution 4.0 International License.

Antibodies Used: Anti-Neuroligin-3 [IPI-mNLGN3.3]
Addgene #219625; Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
Jackson ImmunoResearch #111-606-050

Materials & Methods

Sample

Target Species: Mouse
Cell / Tissue Type: ExpiCHO
Additional Information: ExpiCHO cells were transiently transfected with GFP (transfection control) and V5-tagged mouse NLGN3

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-Neuroligin-3 [IPI-mNLGN3.3]
Source: Addgene
Catalog Number: 219625
Host Species: Rabbit
Target Antigen: NLGN3
Concentration: 1 µg/mL (1:1,000 dilution)
Time: 60 min
Temperature: Room Temperature

View in Addgene Catalog

Secondary Antibody

Name: Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
Source: Jackson ImmunoResearch
Catalog Number: 111-606-050
Concentration: 2 µg/mL
Time: 60 min
Temperature: Room Temperature

Additional Information

Additional Information

Confocal images taken at 40X magnification on an EV0S-m7000 microscope and deconvolved using the richardson-lucy algorithm in the FIJI deconlab plugin.

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-mNLGN3.3 labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 4095 (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 NLGN3.