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ICC/IF for Rabbit IgG Isotype Control [IPI-MOPC-21] in specificity assay

Addgene #237822

Data Submitted By
Alexander Morano , Travis Riedel and Deborah Moshinsky
Lab Name
Institute for Protein Innovation (IPI)
Submission Date
March 25, 2026
Publication Date
March 27, 2026 (modified April 13, 2026)
Abstract
Experimental results for Rabbit IgG Isotype Control [IPI-MOPC-21] (Addgene #237822) in an immunofluorescence (IF) specificity assay with ExpiCHO cells expressing human or mouse V5-tagged ROBO1, CNTN3, NRP1, or GPC1.
Each graph depicts the combined quantification of multiple images of the same transfected cells taken at 10X magnification. GFP- or BFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing IPI-MOPC21 labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 22000. IPI-MOPC21 labeling of human and mouse variants of selected IPI targets from different families is compared on the top and bottom rows. To test cross-reactivity, IPI-MOPC21 was used at 1 μg/mL (1:1000 dilution).
Caption: Each graph depicts the combined quantification of multiple images of the same transfected cells taken at 10X magnification. GFP- or BFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing IPI-MOPC21 labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 22000. IPI-MOPC21 labeling of human and mouse variants of selected IPI targets from different families is compared on the top and bottom rows. To test cross-reactivity, IPI-MOPC21 was used at 1 μg/mL (1:1000 dilution).
Image attribution: Institute for Protein Innovation (IPI)

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

Antibodies Used
Rabbit IgG Isotype Control [IPI-MOPC-21]
Addgene #237822
Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
Jackson ImmunoResearch #111-606-046

Materials & Methods

Sample

Target Species
Human
Cell / Tissue Type
ExpiCHO
Additional Information
ExpiCHO cells were transiently transfected with human or mouse V5-tagged ROBO1, CNTN3, NRP1, or GPC1. Human targets were cotransfected with GFP and mouse targets were cotransfected with BFP (transfection controls).

Buffer

Name
PBS

Fixative

Fixative
Formaldehyde/Paraformaldehyde
Time
15 min
Temperature
Room Temperature

Permeabilization

Agent
Triton X-100 in PBS
Concentration
0.20%
Time
10 min
Temperature
Room Temperature

Blocking

Agent
Normal goat serum + BSA
Concentration
5% serum, 5% BSA
Time
30 min
Temperature
Room Temperature

Primary Antibody

Name
Rabbit IgG Isotype Control [IPI-MOPC-21]
Source
Addgene
Catalog Number
237822
Host Species
Rabbit
Concentration
1 µg/mL (1:1,000 dilution)
Time
60 min
Temperature
Room Temperature
View in Addgene Catalog

Secondary Antibody

Name
Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG Fc fragment
Source
Jackson ImmunoResearch
Catalog Number
111-606-046
Concentration
2 µg/mL
Time
60 min
Temperature
Room Temperature

Additional Information

Additional Information

GFP- or BFP-positive cells were identified via the neural network CellPose, then the mean fluorescence intensity (MFI) of the far-red channel for each cell, representing Rabbit IgG Isotype Control [IPI-MOPC-21] labeling, was recorded. Each histogram displays the number of cells with MFIs ranging from below 100 (background fluorescence) to 22000 (saturation).

Controls

Positive control: Anti-V5 [IPI-SV5-Pk1] (Addgene #218107) used to confirm IF labeling efficacy (since construct expressing target also has a V5 tag.)

Negative control: Rabbit IgG used to confirm lack of nonspecific binding to the sample.

Results

Result
Pass (The antibody worked under these conditions)
Describe Results
The antibody does not show nonspecific binding in the sample or to either of the selected targets