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

Addgene #251720

Data Submitted By: Alexander Morano , Travis Riedel and Deborah Moshinsky
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: February 06, 2026
Publication Date: March 11, 2026 (modified March 13, 2026)
Abstract: Experimental results for Anti-ROBO3 [IPI-ROBO3.119] (Addgene #251720) in an immunohistochemistry (IHC) assay with a mouse spinal cord sample.

**Caption:** Immunohistochemistry (IHC) of a 20 micron cryosection of the spinal cord of an E13 mouse embryo. Confocal images taken at 20X magnification on an ImageXpress confocal HT.ai microscope. Left image shows Anti-ROBO3 [IPI-ROBO3.119] (Addgene #251720) (magenta), commissural axon-labeling counterstain TAG1 (green), and DAPI (blue); the middle image only shows IPI-ROBO3.119, and the rightmost image only shows TAG1. IPI-ROBO3.119 was used at 10 µg/mL (1:100 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-ROBO3 [IPI-ROBO3.119]
Addgene #251720; Goat anti-rabbit alexa fluor 647
ThermoFisher #A21245

Materials & Methods

Sample

Target Species: Mouse
Cell / Tissue Type: Spinal cord
Additional Information: 20 μm section

Buffer

Name: PBS

Fixative

Fixative: Paraformaldehyde, 4% in PBS
Time: 1.5 h post-fixation with PFA after perfusion and harvesting
Temperature: 4 ˚C

Permeabilization / Delipidation

Agent: PBS with 0.1% Triton-X 100
Time: 15 min (3x 5 min washes)
Temperature: Room Temperature

Blocking

Agent: Normal Goat Serum
Concentration: 5% in PBST
Time: 1 h
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)
Time: Overnight
Temperature: 4 ˚C

View in Addgene Catalog

Secondary Antibody

Name: Goat anti-rabbit alexa fluor 647
Source: ThermoFisher
Catalog Number: A21245
Concentration: 1:500 dilution from a 2 mg/mL stock
Time: 90 min
Temperature: Room Temperature

Additional Information

Additional Information

Immunohistochemistry Methods: Mouse embryonic spinal cord

Mouse embryos were first purchased from Jackson Labs. 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 h at 4 °C, then washed 3x 10 minutes in 1X PBS, then stored in 1X PBS + 0.01% sodium azide at 4 °C.

Equilibrated tissue was then frozen in OCT on dry ice and 20–25 μm sections were cut on a Leica cryostat at the Harvard histology/sectioning core. Slides were thawed for 5 min at RT; sections were rehydrated 1x 5min in 1X PBS and then washed 3x 5 min in 1X PBST (0.1% Triton X-100 in 1X PBS) in a humidified chamber in a slide box. Slides were blocked for 1 h at RT in 5% NGS (Normal Goat Serum) or 5% NDS (Normal Donkey Serum) in 0.1% Triton-X-100 in PBS. Primary antibodies were then diluted in blocking solution and incubated for overnight at 4 °C. Slides were then washed 4x 5min in 0.1% TritonX-100 and incubated with secondary antibodies diluted in blocking solution (5% NGS or NDS) at RT for 1.5–2 h, in the dark. Slides were washed 4x 5 min in 0.1% TritonX-100 again, then 1x 5min in 1X PBS. Slides were then mounted in Fluoromount mounting medium with DAPI, outlined with nail polish to secure the coverslip, and stored at 4 °C in the dark until ready to image.

Controls

Positive control: commissural axon-labeling counterstain TAG1.

Negative control: No primary control.

Results

Result: Pass (The antibody worked under these conditions)
Describe Results: This antibody shows strongest labeling along the commissural axons, particularly as they cross the floor plate, in accordance with extant literature describing the localization of ROBO3.