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Anti-ROBO3 [IPI-ROBO3.119] in Immunohistochemistry (Mouse)

Addgene #251720

Data Submitted By: Celine Santiago and Rob Meijers
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: April 01, 2026
Publication Date: April 14, 2026 (modified April 14, 2026)
DOI: https://doi.org/10.57733/addgene.qivt2j
Abstract: Experimental results for Anti-ROBO3 [IPI-ROBO3.119] (Addgene #251720) in an immunohistochemistry (IHC) assay with a mouse embryonic spinal cord frozen tissue section.
Citation: Anti-ROBO3 [IPI-ROBO3.119] in Immunohistochemistry (Mouse). Santiago C, Meijers R 2026. Addgene Report, https://doi.org/10.57733/addgene.qivt2j

**Caption:** Immunohistochemistry of a transverse section of the spinal cord from Embryonic Day 13 (E13) wild type C57BL/6J mouse embryo. 25 micron frozen sections were labeled with Anti-ROBO3 [IPI-ROBO3.119] (Addgene #251720) (10 μg/mL)(Right) and anti-Contactin 2 (4D7/TAG1 antibody from DSHB, 1:100 dilution)(Left) and imaged on an Evos M7000 microscope. Anti-ROBO3 signal is enriched in commissural and pre-crossing axons. Anti-CNTN2 signal is also enriched in commissural and pre-crossing axons, as well as in incoming sensory axons from the dorsal root ganglia. Scale bar = 200 μm.
**Image attribution:** Celine Santiago

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

Antibodies Used: Anti-ROBO3 [IPI-ROBO3.119]
Addgene #251720; 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 section
Preparation: Frozen

Buffer

Name: PBS

Fixative

Fixative: Formaldehyde/Paraformaldehyde
Time: Immediately after harvesting by C-section, fixed whole embryo in 4% PFA in 1X PBS for 2 hours at 4 °C.
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 hour
Temperature: Room Temperature

Primary Antibody

Name: Anti-ROBO3 [IPI-ROBO3.119]
Source: Addgene
Catalog Number: 251720
Host Species: Rabbit
Target Antigen: ROBO3
Concentration: 10 µg/mL (1:100 dilution)
Conjugate: None
Time: Overnight
Temperature: 4 ˚C

View in Addgene Catalog

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

C57BL/6J Embryonic Day (E13) mouse embryos were purchased from Jackson Labs (stock #000664). Instructions were provided to Jackson labs for harvesting and fixing tissue. Immediately after harvesting by C-section, embryos were fixed in 4% PFA in 1X PBS for 2 hours at 4 °C, washed 3x 10 min in 1X PBS, then stored in 1X PBS + 0.01% sodium azide at 4 °C until shipping. Tissue was shipped in 1X PBS + 0.01% sodium azide solution, on cold packs.

Upon receipt of tissue, embryos were washed 3x 10 min in 1X PBS, then cryoprotected overnight in 30% sucrose in 1X PBS, at 4 °C.

Tissue was frozen in Optimum Cutting Temperature (OCT) on dry ice and stored at -80 °C. When ready to section, tissue was brought to -22 °C in cryostat for 1 hour, and 20–25 μm sections were cut on a Leica cryostat at the Harvard Department of Stem Cell & Regenerative Biology Histology core. Slides were stored at -80 °C for 2–3 weeks until ready to label.

On Day 1 of labeling, slides were thawed for 5 min at RT; sections were rehydrated 1x 5 min in 1X PBS and then permeabilized 2x 5 min in 1X PBST (0.1% Triton X-100 in PBS) in a humidified chamber. Slides were then blocked for 1 hour at RT in 5% NGS (Normal Goat Serum) in 0.1% Triton-X-100 in PBS. Primary antibodies were diluted in blocking solution and incubated overnight in the humidified chamber at 4 °C.

On Day 2 of labeling, slides were washed 4x 5 min in 0.1% TritonX-100/PBS and incubated with secondary antibodies diluted in blocking solution (5% NGS/PBST) at RT for 1.5–2 hours, in the dark. Slides were washed 4x 5 min in 0.1% TritonX-100 then 1x 5 min in PBS. Slides were mounted in Fluoromount mounting medium with DAPI (Southern Biotech 0100-2), outlined with nail polish to secure the coverslip, and stored at 4 °C in the dark until ready to image.

Slides were imaged on an Evos M7000 microscope at 10X magnification.

Controls

Positive control: anti-Contactin2 mouse IgM antibody to label commissural axons (DSHB 4D7/TAG1, 1:100)

Negative control: No rabbit primary antibody, all other conditions identical (not shown).

Results

Result: Pass (The antibody worked under these conditions)
Describe Results: This antibody labeled commissural axons as they approach and cross the floor plate; minimal signal on post-crossing axons or in dorsal root ganglia.