Anti-CNTN2 [IPI-CNTN2.57] in Immunohistochemistry (Mouse)
Addgene #254655
- Data Submitted By
- Celine Santiago and Rob Meijers
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- May 21, 2026
- Publication Date
- June 09, 2026 (modified June 09, 2026)
- DOI
- https://doi.org/10.57733/addgene.auk8jd
- Abstract
- Experimental results for Anti-CNTN2 (TAG-1) [IPI-CNTN2.57] (Addgene #254655) in an immunohistochemistry (IHC) application on mouse embryonic spinal cord.
- Citation
-
Anti-CNTN2 [IPI-CNTN2.57] in Immunohistochemistry (Mouse). Santiago C, Meijers R 2026. Addgene Report, https://doi.org/10.57733/addgene.auk8jd
![Immunohistochemistry (IHC) of 20 micron cryosection of the spinal cord of E13 (embryonic day 13) C57BL/6J mouse embryo. Widefield images were taken at 10X magnification on an EVOS-m7000 microscope. Scale bars = 200 μm. Merged image shows signal from Anti-CNTN2 (TAG-1) [IPI-CNTN2.57] (Addgene #254655) (magenta), DSHB 4D7/TAG1 (green), and DAPI (blue). IPI-CNTN2.57 was used at 5 μg/mL (1:200 dilution).
IPI-CNTN2.57 labels sensory axons entering the dorsal horn of the spinal cord, and commissural axons from a subset of spinal cord interneurons, matching the expected localization pattern of Contactin-2 based on published literature.](https://datahub-media.addgene.org/report-images/auk8jd/CNTN2_57_DataSubmission.png)
Image attribution: Celine Santiago
This report is made available under the Creative Commons Attribution 4.0 International License.
- Antibodies Used
-
Anti-CNTN2 (TAG-1) [IPI-CNTN2.57]
Addgene #254655 -
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647
Thermofisher #A-21245
Materials & Methods
Sample
- Target Species
- Mouse
- Cell / Tissue Type
- Mouse E13 spinal cord
- Additional Information
- 20 μm frozen sections on slides
- Preparation
- Frozen
Buffer
- Name
- PBS
Fixative
- Fixative
- Formaldehyde/Paraformaldehyde
- Time
- 2 h in 4% PFA/PBS
- Temperature
- 4 ˚C
- Antigen Retrieval Method
- None
Permeabilization / Delipidation
- Agent
- Triton X-100
- Concentration
- 0.1% in PBS
- Time
- 2x 5 minutes
- Temperature
- Room Temperature
Blocking
- Agent
- NGS (Normal Goat Serum)
- Concentration
- 5% NGS in PBST (0.1% Triton in PBS)
- Time
- 1 h
- Temperature
- Room Temperature
Primary Antibody
- Name
- Anti-CNTN2 (TAG-1) [IPI-CNTN2.57]
- Source
- Addgene
- Catalog Number
- 254655
- Host Species
- Rabbit
- Target Antigen
- CNTN2
- Concentration
- 5 µg/mL (1:200 dilution)
- Time
- Overnight
- Temperature
- 4 ˚C
Secondary Antibody
- Name
- Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647
- Source
- Thermofisher
- Catalog Number
- A-21245
- Host Species
- Goat
- Concentration
- 1:500 dilution from a 2 mg/mL stock
- Conjugate
- Alexa Fluor™ 647
- Time
- 2 hours
- Temperature
- Room Temperature
Additional Information
- Additional Information
Brief Method:
- Obtained mouse tissue from Jackson Labs (After C-section, whole embryos were fixed 2 h in 4% PFA/PBS at 4°C)
- Washed 3x 10 min in 1X PBS, then cryoprotected overnight in 30% sucrose in 1X PBS, at 4°C.
- Froze equilibrated tissue in Optimum Cutting Temperature (OCT) on dry ice, and sectioned tissue on Leica cryostat, 20 microns/section.
- Re-hydrated sections 1x 5min with PBS, then washed and permeabilized (2x 5 min with 0.1% TritonX-100 in PBS, hereafter referred to as PBST).
- Incubated slides in blocking buffer (5% NGS/PBST) for 1 h at room temperature (RT).
- Incubated with primary antibodies overnight at 4°C (5 μg/mL for IPI-CNTN2.57, 1:100 for DSHB 4D7/TAG1), diluted in blocking buffer.
- Washed 4x 5 min with PBST, then incubated with secondary antibodies (goat anti-MsIgM or goat anti-Rabbit, conjugated to Alexa fluorophores) at 1:500 for 2 h at RT in blocking buffer.
- Washed 4x 5 min with PBST, then 1x 5 min in PBS, and mounted with Fluoromount + DAPI.
- Checked signal/imaged slides on Evos M7000 microscope.
- Controls
Positive control: Anti-Contactin-2 mouse IgM antibody to label commissural axons (DSHB 4D7/TAG1, Lot# 12/12/24, 1:100 dilution of 30 µg/mL stock)
Negative control (not imaged): No rabbit primary antibody.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- IPI-CNTN2.57 shows strong and specific labeling of sensory axons entering the dorsal horn of the spinal cord, and of the commissural axons from a subset of spinal cord interneurons, matching the expected localization pattern of Contactin-2 protein.