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IHC for Anti-Netrin-1 [IPI-NTN1.43]

Addgene #241894

Data Submitted By
Samantha Butler and Yesica Mercado-Ayon
Lab Name
Samantha Butler, UCLA
Submission Date
November 14, 2025
Publication Date
November 26, 2025 (modified December 17, 2025)
DOI
https://doi.org/10.57733/addgene.amfit7
Abstract
Experimental results for Anti-Netrin-1 [IPI-NTN1.43] (Addgene #241894) in an immunohistochemistry assay with a rat sample.
Citation
IHC for Anti-Netrin-1 [IPI-NTN1.43]. Butler S, Mercado-Ayon Y 2025. Addgene Report, https://doi.org/10.57733/addgene.amfit7
Rat embryo spinal cord sections (30 µm) were fixed and labeled with Anti-Netrin 1 [IPI-NTN1.43] (Addgene #241894). Labeling of the floor plate coincides with known NTN1 expression patterns.
Caption: Rat embryo spinal cord sections (30 µm) were fixed and labeled with Anti-Netrin 1 [IPI-NTN1.43] (Addgene #241894). Labeling of the floor plate coincides with known NTN1 expression patterns.
Image attribution: Samantha Butler, UCLA

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

Antibodies Used
Anti-Netrin-1 [IPI-NTN1.43]
Addgene #241894
Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L)
Jackson ImmunoResearch #711-605-152

Materials & Methods

Sample

Target Species
Rat
Cell / Tissue Type
Embryo spinal chord
Additional Information
30 µm section
Preparation
Frozen

Buffer

Name
PBS

Fixative

Fixative
Formaldehyde/Paraformaldehyde
Time
60 min
Temperature
4 ˚C

Blocking

Agent
1X PBS, 1% HIHS (heat inactivated horse serum), 0.05% NaAzide, 0.1% Triton-X
Time
30 min
Temperature
Room Temperature

Primary Antibody

Name
Anti-Netrin-1 [IPI-NTN1.43]
Source
Addgene
Catalog Number
241894
Host Species
Rabbit
Target Antigen
NTN1
Concentration
1:200 from 1 mg/mL stock solution
Time
Overnight
Temperature
4 ˚C
View in Addgene Catalog

Secondary Antibody

Name
Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L)
Source
Jackson ImmunoResearch
Catalog Number
711-605-152
Concentration
1:700 dilution from 1.5 mg/mL stock
Time
60 min
Temperature
Room Temperature

Additional Information

Additional Information

PROTOCOL: Immunolabeling of rat and chick embryos

  1. Harvest embryos at desired stages.
  2. MOUNT:
    1. Fix embryos in 4% PFA for 1 h at 4 ºC while rocking.
    2. Wash 3x with 1X PBS then place in 30% sucrose ON.
    3. Next day mount in OCT block and dissect cervical thoracic and lumbar segments of spinal cord:
      1. First, remove intestines and other non-spinal tissues.
      2. Lay spinal cord parallel to surface and with a flat edge razor blade cut clean perpendicular cuts to the spinal.
  3. Cryosection: For axon detection do 30 micron sections, for nuclear markers do 12 micron sections.
    1. LABEL:
      1. Block with PBNT-H 30’ at RT.
      2. 3x 5 min wash with 1X PBS + Tween (0.01%).
      3. Primary antibody O/N at 4ºC.
      4. 3x 5 min wash with 1X PBS + Tween (0.01%).
      5. Secondary antibody 1 h at RT.
      6. 3x 5 min wash with 1X PBS + Tween (0.01%).
      7. MOUNT 65 µl prolong gold (DIMOND), leave 24 h in 4 ºC to solidify.
      8. IMAGE 24 h later.

Horse blocking buffer (PBNTH)
1X PBS, 1% HIHS (heat inactivated horse serum)
0.05% NaAzide
0.1% Triton-X

30% SUCROSE in 0.1 M PB (500 mL)
150 g sucrose
200 mL Na2HPO4 (0.2 M)
47.5 mL NaH2PO4 (0.2 M)
H2O bring to 500 mL Sterile filter and store at 4ºC

Detection Method
Fluorescence

Results

Result
Pass (The antibody worked under these conditions)
Describe Results
Positive labeling for NTN1