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IHC for Anti-Neuroligin-3 [IPI-mNLGN3.18]

Addgene #219629

Data Submitted By
Peter Scheiffele
Lab Name
Peter Scheiffele, University of Basel
Submission Date
October 16, 2025
Publication Date
November 10, 2025 (modified November 11, 2025)
DOI
https://doi.org/10.57733/addgene.w2tpy3
Abstract
Experimental results for Anti-Neuroligin-3 [IPI-mNLGN3.18] (Addgene #219629) in an immunohistochemistry assay with a mouse sample.
Citation
IHC for Anti-Neuroligin-3 [IPI-mNLGN3.18]. Scheiffele P 2025. Addgene Report, https://doi.org/10.57733/addgene.w2tpy3
Representative immunofluorescence images of cerebellar sections from adult C57Bl6/J wild-type and Nlgn3–Stop-tetO knockout mice stained with the monoclonal antibody Anti-Neuroligin-3 [IPI-mNLGN3.18] (Addgene #219629). Mice were perfused transcardially with 9% glyoxal in 8% acetic acid (pH 4) as described by Richter et al. (2018) and Konno et al. (2023). Sections were incubated with primary antibody (1 µg/mL) followed by Cy3-conjugated secondary antibody detection. In wild-type cerebellar tissue, strong punctate immunoreactivity in the inner granular layer is observed, consistent with NLGN3 localization at synapses in mossy fiber glomeruli (Baudouin et al., 2012). In contrast, signal is absent in Nlgn3–Stop-tetO knockout tissue, confirming antibody specificity. Scale bar: 10 µm.
Caption: Representative immunofluorescence images of cerebellar sections from adult C57Bl6/J wild-type and Nlgn3–Stop-tetO knockout mice stained with the monoclonal antibody Anti-Neuroligin-3 [IPI-mNLGN3.18] (Addgene #219629). Mice were perfused transcardially with 9% glyoxal in 8% acetic acid (pH 4) as described by Richter et al. (2018) and Konno et al. (2023). Sections were incubated with primary antibody (1 µg/mL) followed by Cy3-conjugated secondary antibody detection. In wild-type cerebellar tissue, strong punctate immunoreactivity in the inner granular layer is observed, consistent with NLGN3 localization at synapses in mossy fiber glomeruli (Baudouin et al., 2012). In contrast, signal is absent in Nlgn3–Stop-tetO knockout tissue, confirming antibody specificity. Scale bar: 10 µm.
Image attribution: Peter Scheiffele, PhD

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

Antibodies Used
Anti-Neuroligin-3 [IPI-mNLGN3.18]
Addgene #219629
Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)
Jackson ImmunoResearch #711-165-152

Materials & Methods

Sample

Target Species
Mouse
Cell / Tissue Type
Brain (cerebellum)
Preparation
Frozen

Fixative

Fixative
9% Glyoxal in 8% Acetic acid, pH 4.0
Time
24 hr
Temperature
4 ˚C

Permeabilization / Delipidation

Agent
30% Sucrose in 0.1M Phosphate buffer pH 7,4
Concentration
30%
Time
24 hr
Temperature
Room Temperature

Blocking

Agent
10% NGS + 3% BSA + 0.1% Triton X-100
Time
30 min
Temperature
Room Temperature

Primary Antibody

Name
Anti-Neuroligin-3 [IPI-mNLGN3.18]
Source
Addgene
Catalog Number
219629
Host Species
Rabbit
Target Antigen
NLGN3
Concentration
1 µg/mL
Time
24 hr
Temperature
4 ˚C
View in Addgene Catalog

Secondary Antibody

Name
Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)
Source
Jackson ImmunoResearch
Catalog Number
711-165-152
Host Species
Donkey
Concentration
1 µg/mL
Conjugate
Cy3
Time
60 min
Temperature
Room Temperature

Additional Information

Additional Information

References

Richter et al. (2018). Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J, 37(1):139-159. https://doi.org/10.15252/embj.201695709 PMID: 29146773

Konno et al. (2023). Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research. Sci Adv, 9(28):eadf7084. https://doi.org/10.1126/sciadv.adf7084 PMID: 29146773

Badouin et al. (2012). Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism. Science, 5;338(6103):128-32. https://doi.org/10.1126/science.1224159 PMID: 22983708

Detection Method
Fluorescence

Results

Result
Pass (The antibody worked under these conditions)
Describe Results
Strong punctate immunoreactivity in the inner granular layer is observed, consistent with NLGN3 localization at synapses in mossy fiber glomeruli (Baudouin et al., 2012).
Knockout Validation
Yes