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ICC/IF for Anti-Neuroligin-2 [IPI-mNLGN2.4] in siRNA KD assay

Addgene #219624

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.dwifpq
Abstract: Experimental results for Anti-Neuroligin-2 [IPI-mNLGN2.4] (Addgene #219624) in an immunofluorescence (IF) assay with U-188 MG cells treated with siRNA against NLGN2.
Citation: ICC/IF for Anti-Neuroligin-2 [IPI-mNLGN2.4] in siRNA KD assay. Morano A, Riedel T, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.dwifpq

**Caption:** Immunofluorescence (IF) of U118-MG cells treated with 20 nM siRNA against housekeeping gene cyclophilin B (top row) and 20 nM siRNA against NLGN2 for 90 hours, then labeled for NLGN2 expression using IPI-mNLGN2.4 and for actin with Alexa Fluor 488-conjugated phalloidin. Confocal images taken at 40X magnification on an ImageXpress HT confocal.ai microscope. Left images show all 3 channels (IPI-mNLGN2.4, actin, and DAPI) while the right images only show IPI-mNLGN2.4 and DAPI. For both panels, IPI-mNLGN2.4 was used at 5 µg/mL (1:200 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-2 [IPI-mNLGN2.4]
Addgene #219624; 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: U-188 MG
Additional Information: U-188 MG cells were treated with siRNA against housekeeping gene cyclophilin B (Horizon Research #D-001820-10-05) or against NLGN2 (Horizon Research #L-008843-01-0005).

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-2 [IPI-mNLGN2.4]
Source: Addgene
Catalog Number: 219624
Host Species: Rabbit
Target Antigen: NLGN2
Concentration: 5 µg/mL (1:200 dilution)
Time: 60 min
Temperature: Room Temperature

View in Addgene Catalog

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

As a control, the same experiment was performed using scrambled siRNA or siRNA targeting the housekeeping gene cyclophilin B, and resulted in no significant change in labeling (data not shown).

The antibody used for cyclophilin B detection was Fisher Scientific PA1-027A.

Results

Result: Pass (The antibody worked under these conditions)
Describe Results: siRNA knockdown resulted in reduced labeling with IPI-mNLGN2.4 by a significant degree in cells expressing NLGN2 using a siRNA specific to NLGN2 but not with control siRNA.
Knockout Validation: Yes